Fluorescent dyes (AIDA) for solid phase and solution phase screening

ABSTRACT

The invention relates to new fluorescent dyes of formula (I)  
                 
which can be used in high throughput screening both, on the solid phase as well as in homogeneous solution.

TECHNICAL FIELD

The present invention relates to the field of ultra high-throughputscreening on the solid support and in homogeneous solution by a novelgeneric labelling technology. The new labelling technology is based onnew chemically stable fluorophores, which possess reactive chemicalfunctionalities for attachment to a solid support and subsequent startof combinatorial synthesis of compound libraries.

BACKGROUND

Three new scientific disciplines show the highest promise of fulfillingthe need for increased predictability and for lowering the overallattrition rate of the drug discovery process. (1) Functional Genomicswas invented to generate new innovative molecular targets. (2)Combinatorial Chemistry provides increasingly efficient ways to generatemolecular diversity with which to probe the targets. (3) High-throughputscreening (HTS) platforms provide efficiency and quality in findingpotential lead compounds. High throughput screening within mostpharmaceutical companies currently involves performing several millionassays per year. Meanwhile, HTS has become a discrete disciplineassimilating biochemistry, biophysics and cell/molecular biologycombined with detection/liquid handling technologies and automationprocesses. With all the phantastic oportunities these new scientificdisciplines offer, it became already clear, that the time consumingprocess in functional genomics is the identification of thephysiological function of a new protein. Whereas combinatorial chemistryapproaches help to synthesize a multifold of compounds during the timeneeded for classical synthesis, it is known now that between 5 and 10times the number of assays are needed to identify hit compounds fromscreens. The challenge the community of applied science is currentlyfaced with, is to fully exploit the advantages of (1) and (2) in atimely manor by speeding up the process of synthesis, protein functionidentification and screening. The current invention opens a newpossibility for integrating the advantages of combinatorial chemistryand genomics with HTS by providing the efficiency needed for screeningcompounds directly on the solid support. Target macromolecules of evenunknown functionality can be tested for their direct binding affinity tocompounds of interest. The new fluorescent chemistry, genericallydescribed as AIDA-chemistry in the following, is suitable also toscreening assays in homogeneous solution, either by direct applicationof the compounds conjugated to the AIDA chemistry or by cleavage of theAIDA conjugates from the solid support by well known chemical orphotophysical means. After the release of the AIDA conjugated “binder”identified in a solid-phase screening technology, the affinity to themacromolecule of interest can be determined by conventional ensembleaveraging fluorescence spectroscopic techniques in assay volumes used inmicrotiter plates. In addition, single molecule spectroscopic techniquesperformed in microliter volumes and applied in so called nanocarrierscan be used.

SUMMARY OF THE INVENTION

The invention refers to specific fluorescent dyes, which can be used inhigh throughput screening both, on the solid phase as well as inhomogeneous solution. The new fluorescent dyes generically referred toas AIDA chemistry is suitable for various methods of solid phase andsolution phase organic chemistry for synthesis of molecules to beinvestigated for therapeutic use in disease states. As used herein,“AIDA” is an abbreviation for arylindazol compounds or derivativesdescribed herein. The molecules of therapeutic interest can besynthesized as fluorescent conjugates by two methods: (a) a solidsupport is loaded with a cleavable linker (acid-, base-, redox- or lightsensitive) to which initially the fluorescent dye is attached. The dyespossess a second functionality, which serves as attachment point forspacer elements,. The spacer bears a further functional group which isused as starting point of the synthesis of the molecules to beinvestigated; (b) the fluorescent dye can also be introduced as end-capin the last synthesis step of a reaction sequence. The specific dyesdescribed in the invention are chemically stable under a broad range ofreaction conditions usually applied in solid phase and solution phaseorganic chemistry. The conjugates emit fluoresence in the visible andUV-spectrum on excitation at wavelengths of their absorption. Thesefluorescence properties allow for multiple applications in fluorescencebased processes for the identification of inhibitors of molecularinteractions and for the identification of molecules which bind totarget macromolecules like peptides proteins, nucleic acids,carbohydrates etc. The fluorescence detection technologies used formonitoring binding of AIDA-conjugated compounds to macromoleculesinclude conventional macroscopic techniques (ensemble averaging),whichdetect changes in fluorescence intensity, anisotropy(polarization),fluorescence resonance energy transfer, fluorescence lifetime,rotational correlation time as well as single molecule spectroscopictechniques (SMS). SMS include excitation by one or two laser wavelengthsincluding laser lines at 325 nm, 351 nm, 453 nm, 488 nm, 514 nm, 543 nm,632 nm and other excitation possibilities. The fluorescence lightemitted from single molecules diffusing through a confocal focus asapplied in single molecule spectroscopy passes through one or twofilters, and polarisers before the light reaches the avalanchephotodiode detector. The comprehensive detection technologies in SMS tobe applied to AIDA include translational diffusion, rotationaldiffusion, fluorescence lifetime, fluorescence brightness, spectralshifts, fluorescence energy transfer, triplet transition probabilitiesand multiplex detection. The dyes show sufficient stability in chemicaltransformations, have little triplet state formation and are notphotoreactive under the conditions used for detection of binding eventsto biomolecules, making them to an excellent tool for combination ofcombinatorial chemistry (solid phase and solution phase chemistry) andbiological investigations (ultra high throughput screening).

Uses of the dye include solid phase and solution phase organicchemistry, low molecular weight compound labelling, peptide labelling,protein labelling, optical spectroscopy and fluorescence. Synthesis offunctionalized dyes and of dye conjugates (on solid support and insolution) are disclosed.

DETAILED CHEMICAL ASPECTS OF THE INVENTIONS

A first aspect of the invention is directed to a fluorescent dyerepresented by formula (I)

Wherein one of the radicals R¹ or R² and one of the radicals R³ or R⁴ ishydrogen and the other is independently —COOH, —COOR⁷, —CONH₂, —CONR⁸R⁹,—CONH(CH₂)_(n)OH, wherein n=2-8, —CH₂OH, —CH₂NH₂, —NO₂, NR¹⁰R¹¹,NHCOR¹², Cl, Br, F, —CF₃, O(C₁-C₄)-alkyl (optionally substituted bymethyl or phenyl at any of the carbons C₁-C₄), —N═C═O, N═C═S, —SO₃H,—SO₂NH(CH₂)_(n)NH₂, (C₁-C₄) alkyl, (C₁-C₁₆)-alkyl substituted at theterminal carbon with —COOH, —COOR⁷, —CONH₂, —CONR⁸R⁹, —CONH(CH₂)_(n)OH,wherein n=2-8, —CH₂OH, —CH₂NH₂—N═C═O, N═C═S, —SO₃H, —SO₂NH(CH₂)_(n)NH₂,—CONH(CH₂)_(n)NH₂, wherein n=2-8, and the NH₂-group could also besubstituted by (C₁-C₄) alkyl or a commonly used amino protecting groupsuch as tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, phthalimido,trifluoroacetamido, methoxycarbonyl, ethoxycarbonyl, benzyloxycarbonyl,allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl,2-(trimethylsilyl)ethoxycarbonyl,

-   -   and one of the radicals R⁵ or R¹ is hydrogen and the other is        hydrogen, halogen, O(C₁-C₄)-alkyl (optionally substituted by        methyl or phenyl at any of the carbons C₁-C₄), —NO₂, NR¹⁰R¹¹,        NHCOR¹², (C₁-C₄) alkyl, (C₁-C₁₆)-alkyl substituted at the        terminal carbon with —COOH, —COOR⁷, —CONH₂, —CONR⁸R⁹,        —CONH(CH₂)_(n)OH, wherein n=2-8, —CH₂OH, —CH₂NH₂, —N═C═O, N═C═S,        —SO₃H, —SO₂NH(CH₂)_(n)NH₂, —CONH(CH₂)_(n)NH₂, wherein n=2-8, and        the NH₂-group could also be substituted by (C₁-C₄) alkyl or a        commonly used amino protecting group such as        tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, phthalimido,        trifluoroacetamido, methoxycarbonyl, ethoxycarbonyl,        benzyloxycarbonyl, allyloxycarbonyl,        2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl    -   R⁷ is a commonly used carboxyl protecting or carboxyl activating        group, such as succinimidyl, azido, phenyl, 4-nitrophenyl, and        pentafluorophenyl for activation and methyl, β-substituted        ethyl, 2,2,2-trichloroethyl, tert-butyl, allyl, benzyl,        benzhydryl, 4-nitrobenzyl, 2-(4-toluenesulfonyl)ethyl, silyl,        2-(trimethylsilyl)ethyl, MEM, MOM, BOM, MTM, and SEM for        protection, respectively.    -   R⁸ or R⁹ is hydrogen and the other is lower alkyl (C₁-C₄),        phenyl, benzyl, or R⁸ and R⁹ are part of a 5 or 6 membered such        as in piperazine    -   R¹⁰ and R¹¹ are independently hydrogen, (C₁-C₄) alkyl    -   R¹² is (C₁-C₁₀) alkyl, phenyl, which both can be substituted by        (C₁-C₄) alkyl, protected (suitable protecting groups are        mentioned above) amino group or halogen.

Preferred embodiments of these aspects are fluorescent indazole dyesrepresented by the following structures:

Another aspect of the invention is directed to fluorenscent conjugatesrepresented by formula (II-III)A-B-D-C-D′-E   (Formula II))A-B-D-E-D′-C   (Formula (III))wherein

A is a solid support selected from standard materials applied in solidphase and solution phase organic chemistry (e.g. functionalizedpolystyrene based resins, polyacrylamide based polymers,polystyrene/polydimethylacrylamide composites, PEGA resins,polystyrene-polyoxyethylene based supports, Tentagel, PEG-polystyrenegraft polymeric supports, glass surfaces, functionalized surfaces,materials grafted with functionalized surfaces, or polyethylenglycol).

-   -   B is a linker allowing cleavage of fluorescent conjugates of        formula (II-III) for liberation of the D-C-D′-E or D-E-D′-C        fragment, respectively. B is selected from the known acid        labile, base labile, light labile, redox-labile, and masked        linkers applied in combinatorial synthesis, peptide synthesis,        and oligonucleotide synthesis (e.g. benzyl, benzhydryl,        benzhydryliden, trityl, xanthenyl, benzoin, silicon, or allyl        based linkers).

C is a compound selected from formula (I)

D and D′ are independently a bond or a spacer selected fromα,ω-diamino-alkanes, diaminocyclohexyl, bis-(aminomethyl)-substitutedphenyl, α-amino-ω-hydroxy-alkanes, alkylamines, cyclic alkylamines,cyclic alkyldiamines or amino acids without or with additionalfunctionality in the side chain.

E is the molecule to be investigated e.g. a low molecular weightcompound, a peptide, a protein, a carbohydrate, a nucleic acid, or alipid bearing a functional group for attachment.

Conjugates of formula (II) can be generated via two independentprotocols: (a) by de novo synthesis of molecule E, such incorporating afunctional group of the spacer D′ of the A-B-C-D′ or A-B-D-C-D′fragment, respectively. (b) by attachment of a pre-built molecule Econtaining a suitable functional group for coupling to the A-B-C-D′ orA-B-D-C-D′ fragment, respectively (e.g. an amino or carboxy terminus).Conjugates of formula (III) can be generated either by de novo synthesisof molecule E starting with the A-B or A-B-D conjugate, and subsequentcapping with a D′-C or C fragment, or by attachment of a pre-builtmolecule E containing suitable functional groups for coupling to the A-Bor A-B-D, and D′-C or C fragment, respectively (e.g. an amino andcarboxy terminus).

Preferred compounds of formula (I) used as C for synthesis of conjugatesrepresented by formula (II) are:

Preferred compounds of formula (I) used as C for synthesis of conjugatesrepresented by formula (III) are:

Preferred conjugates of formula II are represented by the followingstructures:

Preferred conjugates of formula III are represented by the followingstructures:

A further aspect of the invention is directed to AIDA conjugatesrepresented by formula (IV):E-D′-C   (Formula (IV))wherein

E is the molecule to be investigated e.g. a low molecular weightcompound, a peptide, a protein, a carbohydrate, a nucleic acid, or alipid bearing a functional group for attachment.

D′ is a bond or a spacer selected from α,ω-diamino-alkanes,diaminocyclohexyl, bis-(aminomethyl)-substituted phenyl,α-amino-ω-hydroxy-alkanes, alkylamines, cyclic alkylamines, cyclicalkyldiamines or amino acids without or with additional functionality inthe side chain.

C is a compound selected from formula (I).

Preferred compounds of formula (I) used as C for synthesis of conjugatesrepresented by formula (IV) are as given for formulas II and III.

Preferred compounds of formula (IV) are represented by the followingstructures:

DETAILED METHODOLOGICAL/TECHNOLOGICAL ASPECTS OF THE INVENTION

Among the many feasibly applications based on AIDA dye molecules, themost promising detection procedures are the following:

(1) AIDA molecules on the solid support cannot be excited tofluorescence by the conventional Ar-ion and HeNe laser lines starting at453 nm and predominantly using 488 nm, 514 and 543 nm wavelengths toexcite dye molecules like fluoresceins or rhodamines etc. AIDA dyes cantherefore be used as “silent” fluorescence markers on the solid supportwithout interfering with any kind of detection technology using red(>453 nm) fluorescence.

(2) Once cleaved from the solid support or used on soluble compounds,directly or after cleavage, the AIDA marker can be excited with aUV-laser at 325 or 351 nm excitation source to be detected by variousmethods used in fluorescence spectroscopy.

(a) In conventional cuvettes used for fluorescence experiments or inmicrotiterplates, the binding of AIDA-containing compounds to targetmacromolecules can be detected by the reduction in rotational frequency.The detection parameter applied is the rotational correlations time intime-resolved fluorescence equipments (using the single-photon-countingor phase domain detection modes) or by anisotropy/polarisation incontinous wave=prompt=steady state fluorometers.

(b) In single molecule fluorescence experiments the reduction intranslational diffusion time determined from autocbrrelationcalculations on the time trace of fluorescence fluctuations is theanalogous detection parameter.

(3) With excitation wavelengths ranging approximately from 300 nm to 400nm the emission wavelengths of AIDA dyes are usually very broad reachingfrom aproximately 350 nm to 600 nm. It is well known in the literaturethat UV-excitable dyes show strong environmental sensitivity. By varousdifferent kinds of photophysical interactions with binding partners likeproteins the fluorescence emission of these dyes including AIDA showquenching or enhancement of fluorescence by various different mechanismsincluding charge transfer and electron transfer mechanisms. This resultsin a change of quantum yields or a shift of the spectra to longer orshorter wavelengths. These signal parameters are used to monitor bindingreactions.

(4) For applications with AIDA based dyes another suitable interactionparameter is fluorescence resonance energy transfer (FRET). The rangeand the shape of the absorption spectra of the stable AIDA conjugates(on solid support as well as in solution) show an excellent spectraloverlap with the emission of the fluorescent amino acid tryptophan inproteins. The emission spectra of the AIDA-conjugates (usually between350 and 600 nm) overlap with various conventionally used fluorescencelabels of proteins (e.g. BODIPY, Rhodamine, Fluorescein, Cy). Thesespectral properties enable detection of binding events betweenconjugates and biomolecules (labelled or unlabelled) by fluorescenceresonance energy transfer from excited tryptophan (donor) to the dyemolecule (acceptor) of the conjugate. In a different experimental setup,the fluorescence resonance energy transfer can be established betweenthe AIDA-containing molecule and a fluorescence label (acceptor)conjugated with the biomolecule on excitation of the AIDA (donor). Insum, in macroscopic fluorescence experiments (averaging the signals ofmany molecules in a cuvette or micro-titer plate), AIDA-conjugates canbe used as fluorescence donors or as-fluorescence acceptors in FRETexperiments on the solid support or in homogeneous solution. Complexformation with tryptophan and/or tyrosine containing proteins can bedetected by donor (tryptophan) quenching or acceptor (AIDA)sensitisation. Complex formation with fluorescently labelledmacromolecules (labeles include all dyes excitable to fluorescencebetween 350 nm and 680 nm) can be detected by donor (AIDA) quenching andacceptor (emission energies lower than at 350 nm) sensitisation.

(5) In SMS AIDA-conjugates can also be used as donors and acceptors. TheFRET derived quenching or enhancement of the tryptophan, AIDA or redacceptor dye (e.g. Rhodamine) is monitored by the change in molecularbrightness. UV-excitation of tryptophans, however is only possible withtwo- or three photon excitation by pulsed lasers in the femtosecondfrequency range, wheras AIDA can be excited by cw-UV-lasers at 325 nm or351 nm.

(6) The following assay detection schemes are possible with AIDA andSMS.

(6.1) On the solid support:

(a) Silent AIDA-1: The direct detection of binding of fluorescentlylabelled macromolecules to AIDA containing solid supports applyingconfocal microscopic techniques like the one described in WO 95/35492(Eigen, M. & Henco, K., Process and device for selectively extractingcomponents from complex mixtures).

(b) Silent AIDA-2: With fluorescently tagged solid supports containingAIDA on linked compounds the procedure described in 6.1.a. can beapplied using two excitation and two detection channels. Whereas the tagon the solid support (e.g. in the interior of a bead) can be excited at543 nm and detected at 570 to 580 nm, the target macromolecule which istested for binding to the AIDA-containing compound on the solid supportcan be excited at 632 nm and detected e.g. between 670 and 690 nm. Thisdual color excitation and emission method can strongly increase thespecificity of the detection of the complex formation on the solidsuport by cross-correlating the emission signals of two dyes or bymonitoring the coincedence of the two colors on a detection timescalewithin the scanning confocal focus.

(c) Including AIDA fluorescence-2: The change in molecular brightnesscan be enhanced by chemically linking AIDA to a second environmentallysensitive molecule as commonly used in conventional fluorescencespectroscopy performed during the synthesis of the compound on the solidsupport.

(d) Including AIDA fluorescence-3: As generally described in (5), FRETfrom AIDA to a suitable long wavelength dye which will thereby besensitized using AIDA UV-excitation can be detected by change inmolecular brightness at the emission wavelength of the long wavelengthdye. This acceptor sensitization must by photophysical rules include aquenching of the donor signal. The reduction of specific brightness at351 nm excitation and 400 nm emission wavelengths can therefore beapplied as well for monitoring binding reaction with FRET suitablelabelled macromolecules.

(e) As changes in molecular brightness of a molecule are very oftenconnected with a change in quantum yield, all the potential possibleapplications monitoring changes in molecular brightness are detectablealso in a time-resolved mode in SMS.

(6.2) In homogeneous solution:

Generally all the single molecule spectroscopic detection technologiesdescribed in 6.1. can also be applied in solution after cleavage of theAIDA conjugate from the solid support with the exception of 6.1-a.

The invention therefore relates also to a method for identification ofan interaction between an AIDA labelled molecule and a binding moleculein homogeneous solution wherein the method comprises the followingsteps:

Step 1A: Providing an AIDA labelled molecule selected from formula (IV)

Step 1B: Admixing the AIDA-labelled molecule of formula (IV) with abinding molecule; and then

Step 1C: selectively detecting a binding event with the AIDA-labelledmolecule described in Step 1 B and the binding molecule by methods offluorescence spectroscopy.

The methods of fluorescence spectroscopy are measurements of

-   -   Increase of fluorescence anisotropy/polarisation of AIDA        emission in continuous wave=prompt=steady state fluorometers,    -   Increase of rotational correlation time in time-resolved        fluorescence equipments,    -   Increase in translation diffusion time in single molecule        fluorescence experiments determined from autocorrelation        calculations on the time trace of fluorescence fluctuations,    -   Increase or decrease of AIDA fluorescence emission in the        wavelength range between 350 and 700 nm with excitation        wavelengths in the range between-300 and 400 nm,    -   Fluorescence resonance energy transfer (donor quenching or        acceptor sensitisation) from excited tryptophan (donor) in the        binding molecule which in this case is a peptide or protein to        the AIDA dye (acceptor) in the molecule of the conjugate,    -   Fluorescence resonance energy transfer (donor quenching or        acceptor sensitisation) from the excited AIDA dye in the        conjugate molecule (donor) to a fluorescent label (acceptor) of        the binding molecule which in this case can comprise any        compound class.

Furthermore the invention relates to a method for identification of aninteraction between an AIDA labelled molecule on the solid support whichis conventionally used in solid phase organic chemistry and a bindingmolecule in homogeneous solution containing the solid support whereinthe method comprises the following steps:

Step 2A: Providing an AIDA labelled molecule as conjugate of formula (IIor III)

Step 2B: Admixing the AIDA-labelled molecule as conjugate of formula (IIor III) with a binding molecule; and then

Step 2C: selectively detecting a binding event with the AIDA-labelledmolecule described in Step 2B and the binding molecule by methods usedin fluorescence spectroscopy resulting in a quantitative signalproviding a means to identify the AIDA-linked molecule with the highestbinding affinity to the binding molecule,

Step 2D: Isolation of the solid support containing the identifiedAIDA-molecule represented by formula (II or III)

Step 2E:: Selectively detecting a binding event with the AIDA-labelledmolecule described in Step 2D and the binding molecule by variousmethods used in fluorescence spectroscopy described in the procedure1A-C.

The fluorescence spectroscopic methods in step 2C are

-   -   Direct detection of binding of fluorescently labelled        macromolecules to AIDA containing solid supports applying        confocal microscopic and spectroscopic techniques    -   measurement of enhancement of the change in molecular brightness        by chemically linking AIDA to a second environmentally sensitive        molecule as commonly used in conventional fluorescence        spectroscopy performed during the synthesis of the compound on        the solid support,    -   measurement of fluorescence resonance energy transfer: From AIDA        to a suitable long wavelength dye which will thereby be        sensitised using AIDA UV-excitation detected by change in        molecular brightness at the emission wavelength of the long        wavelength dye,    -   measurement of fluorescence resonance energy transfer: Reduction        of specific brightness of AIDA on the molecule linked to the        solid support at 351 nm excitation and 400 nm emission        wavelengths,    -   Detection of the change in quantum yield by measuring reduction        or increase in molecular brightness by time-resolved single        molecule spectroscopy.

Positioning of the Disclosed AIDA Chemistry Relative to Known IndazoleChemistry and Photophysics.

The invention is directed to fluorescent dyes represented byfunctionalized 1,3-diaryl-1H-indazoles (AIDA). The present inventionallows the synthesis of combinatorial compound libraries with inherentfluorescence label on solid support and in solution. Such labels arepowerful tools for detection of binding events of synthesized ligands tobiomolecules. Binding events between synthesized molecules andbiological targets can be measured by spectroscopic methods as describedin the previous section. These experiments can be performed with dyeconjugates immobilized on a solid support or in solution.

The molecules described in the invention fulfil several chemical andspectroscopic requirements for use in the disclosed applications. Theparent compound of the disclosed indazole based dyes, 1H-indazole, is afluorescent molecule with a maximum of absorption at 294 nm and amaximum of fluorescence emission at 298 nm (both values in acetonitrile;Saha, S. K. & Dogra, S. K. J. Photochem. Photobiol., A (1997), 110,257-266). These spectroscopic properties are not in agreement with therequirements, which a dye has to fulfil for use in the applicationsdescribed above. Only when the indazole chromophore is extended byappropriate substitution with arylsubstituents (which bear additionalfunctional groups for the synthetic applications of the dyes, as suchalso contributing to further extension of conjugation) the necessarybathochromic shifts of the absorption maximum as well as of thefluorescence emission maximum can be achieved to provide for theapplications described in the former sections. The mentioned syntheticmodifications on the parent heterocycle establish the spectral overlapof absorption of the 1,3-diaryl-1H-indazoles (range of absorption maxima326-361 nm for compounds 4a-l, 8, and 11, respectively) with theemission of tryptophan. Not all modifications with regard to extensionof the chromophore of the indazole are beneficial: further extension ofthe fluorophore represented by the 1,3-diaryl-1H-indazoles with anadditionally annellated benzene ring on the heterocycle corroborates thespectral requirements. The absorption spectrum of4-(3-phenyl-benzo[g]indazol-1-yl)-benzoic acid changes to a naphthalenetype spectrum, and the fluorescence emission is dramatically reduced.

The 1,3-diaryl-1H-indazoles show Stokes shifts in the range of 60-200nm, depending on the substitution pattern, and are characterized by avery broad emission band. Emission maxima are in the range 364-439 nmfor derivatives 4a-e, 4g-h, 8, and 11, respectively, and 524-565 nm forderivatives 4f, and 4i-l, respectively, indicating the broadpossibilities for tuning the photophysical properties of such asubstituted 1,3-diaryl-1H-indazole structure. A detailed spectroscopiccharacterization of a representative AIDA-derivative (4e; FIG. 1; forstructure: scheme 1) is described in example 1A. The spectroscopicvariability allows for the specific design of interaction or bindingassays with labelled or unlabelled target macromolecules in solution andon the solid support. The variability in spectroscopic propertiescovering an unusual broad range of fluorescence emission wavelengthswith an absorption wavelength in the gap region between natural proteinfluorescence and conventional dyes used in fluorescence spectroscopy forHTS allows for a unique combination of assay technology, namely (a) fastand efficient screening for binders on the solid support (silent AIDAdye which does not show fluorescence) and (b) immediate confirmation ofthe identified active hit compounds after cleavage from the support bydifferent possible applications of AIDA fluorescence.

One aspect of the invention, as demonstrated in examples 2A and 3A,below, describes the methods used to show binding of a protein (avidin)to its ligand (biotin) conjugated with a dye molecule of the invention.

The conjugate 11 is used to demonstrate the binding of 11 to avidin bythe change of the anisotropy value of free conjugate upon binding to theprotein (example 2A). The tight binding of the AIDA-biotin conjugate 11to avidin is also confirmed by FRET from tryptophans contained in avidinto the AIDA dye, and from the AIDA dye to a BODIPY label on avidin.

Conjugate 8 is synthesized on solid support (example 3B(a)). Theemission spectrum of immobilized 8 resembles the one in solution.AIDA-conjugate 8 is cleaved from the resin, and the FRET fromtryptophans in avidin, as well as from AIDA to BODIPY on avidin isdemonstrated (example 3A). The energy transfer between the AIDA moietyin 8 and the BODIPY fluorescence label on avidin is also studied bytime-resolved fluorescence spectroscopy.

Example 2A and 3A exemplify the use of AIDA dyes to report bindingevents between AIDA-conjugates (8, 11) and unlabelled or labelledbiomolecules (avidin, avidin-BODIPY-FL).

To this end, derivatives of 1,3-diaryl substituted 1H-indazoles (4a-l,5, 6; examples 1B and 2B; schemes 1 and 2) are synthesized possessing(1) a functional group for (a) attachment of the dye to a solid supportpreloaded with a cleavable linker, or (b) to label a compound library bycapping reaction with an appropriate AIDA-derivative, and (2) anadditional functional group for conjugation with the ligand. The latterfunctionality is presented as part of a (variable) spacer elementinserted between dye and ligand. The spacer itself is covalently linkedto the dye by an amide bond. Among the numerous known possibilities tosynthesize the indazole heterocycle (for a review see: Elderfield, R. C.in “Heterocyclic Compounds” ed. Elderfield, R. C., vol 5, John Wiley &Sons, Inc., New York, 1957, p. 162), and more specifically1,3-diaryl-1H-indazoles, the methods described by Gladstone et. al.(Gladstone, W. A. F. & Norman, R. O. C. J. Chem. Soc. (1965), 3048-52;Gladstone, W. A. F. & Norman, R. O. C. J. Chem. Soc. (1965), 5177-82;Gladstone, W. A. F. et. al. J. Chem. Soc. (1966), 1781-4) enable anefficient access to the desired compounds. The synthesis starts fromsubstituted benzophenones. One of the aromatic substituents of theketone is finally incorporated into the indazole skeleton, whereas thesecond one serves as the source for the functionalized 3-arylsubstituentof the indazole. The benzophenone derivatives are first converted toarylhydrazones by reaction with substituted arylhydrazines. The latterare the source for the functional group attached on the1-arylsubstituent of the final, functionalized 1,3-diaryl-indazole. Thebenzophenone arylhydrazones are reacted to acetic acid1-(arylazo)-1,1-diarylmethyl esters by oxidation with lead tetraacetate,following a procedure first described by Iffland (Iffland, D. C. et al.(1961) J. Am. Chem. Soc. 83, 747). The arylazo-acetates undergoring-closure to indazole heterocycles upon treatment with Lewis acids.The reaction proceeds via an intermediate 1-aza-2-azoniaallene (Wang, Q.et. al. Synthesis (1992), 7, 710-8) and is highly regioselective for thegeminal arylsubstituents of the arylazo-acetates competing forring-closure. Cyclization does not occur into aromatic ringsbearing-strong electron withdrawing substituents, such as carboxyl-,ester-, amide-, or nitrogroups, respectively. Substitution of the arylrings, either in position 1 or position 3, of the indazole by anitro-substituent causes a bathochromic shift of the emission maximum ofabout 150 nm compared to other substitution patterns investigated (table1). The nitro induced red shift of the emission of AIDA-derivativesexpands the spectroscopic applicabilities described above. The sameholds for a nitro-group attached directly to the nucleus of theheterocycle, but such indazole derivatives are not accessible by thediscussed Gladston indazole synthesis, for the reasons given. 5-nitrosubstituted, functionalized 1,3-diaryl-1H-indazoles can be synthesizedby careful nitration of a corresponding indazole as it is exemplified inexample 1B(b). Functionalized 1,3-diarylsubstituted indazoles can alsobe synthesized on solid support starting with immobilized arylhydrazonesof appropriate benzophenone derivatives (Yan, B. & Gstach, H.Tetrahedron Lett. (1996), 37(46), 8325-8).

Specifically, an example 3B(a) describes the principle of solid phasesynthesis of fluorescent conjugates as represented by formulas (II) and(IV). The spacer bearing AIDA-dye (described in example 2B(b)) is firstattached to a linker on a solid support, followed by coupling of theligand, and subsequent cleavage of the AIDA-ligand conjugate (8).

AIDA conjugates as represented by formula (IV) can also be synthesizedin solution as it is demonstrated in example 4B(a-c). Synthesizedcompounds 8 and 9 differ not only in the nature of the spacer introducedfor coupling of the ligand (biotin) to the AIDA dye (4e), but also withregard to the arylsubstituents of the indazole. The conjugate to biotinis built up via the 1-arylsubstituent in derivative 8, whereas theligand is presented on the 3-arylsubstituent in derivative 11. Bothgeometries can be used for detection of binding events (pseudosymmetryof the AIDA-dyes). The principle of the synthesis of compound librariesof fluorescent conjugates as represented by formulas (Ill) and (VI) aredescribed in examples 5B and 7B. The AIDA-dye (4c) was introduced in thefinal step of a reaction sequence, followed by cleavage of theAIDA-ligand conjugates 12a-j, and 14, respectively.

The synthesis of a compound collection of sulfonamides conjugated withan AIDA-dye (13a-d) represented by formulas (II) and (IV) is describedin example 6B.

Experimental Protocols

Part A: Spectroscopic

General

Reagents and Proteins: 10 mM and 5 μM stock solutions of 4e, 9 and 11are prepared in THF (4e) or DMSO and kept at 4° C. avidin andavidin-conjugates are purchased from Molecular Probes (Eugene, Oreg.):A-2767, unlabelled; A-883, Lucifer Yellow (LY); A-2641, BODIPY FL.

200 μM stock solutions are prepared in PBS, 0.01% NaN₃, or 0.1 M NaHCO₃,pH=8.3 (A-2641) and stored at −20° C. Concentrations were determinedgravimetrically assuming a molecular weight of 67000 for avidin andconjugates. For solubility reasons, 4e is spectroscopicallycharacterized in THF. All complexation reactions and fluorescenceresonance energy transfer measurements are performed in PBS/5% DMSO at25° C., unless indicated otherwise.

UV-VIS Spectroscopy:

UV-Vis absorption spectroscopy is performed on a Cary 1Espectrophotometer. The absorption spectrum of 4e is detected in THF.

Determination of the extinction coefficient (ε) of 4e at the absorptionmaximum at 337 nm:

Three different amounts of the sample (E1=5.751 mg, E2=7.933 mg,E3=10.933 mg) are dissolved in tetrahydrofuran (THF, forUV-spectroscopy, Fluka) to a final volume of 25 mL. This results inconcentrations of 577.5 μM, 796.6 μM and 1.0979 mM for E1, E2 and E3,respectively. From each stock solution, 5 dilutions with concentrationsof 2.5 μm, 10 μM, 17 μM, 25 μm and 35 μM are made. UV-spectra of eachdilution are recorded from 200 to 400 nm, using a quarz cuvette with apathlength of 1 cm. THF is used as a reference. A linear fit is carriedout for each series of measurements. The extinction coefficient ε at 337nm is given by the slope of the linear regression.

Steady-State Fluorescence Spectroscopy:

Steady-state fluorescence measurements are performed on a SLM 8000Cspectro-fluorometer equipped with JD490 photomultipliers and a 450 WXenon Arciamp (SLM Instruments, Urbana, Ill.).

Steady-State Fluorescence Spectroscopy of 4e:

Steady state fluorescence measurements are carried out intetrahydrofuran (THF) at 1 μM sample concentration. Spectral bandwidthsare set to 1 and 4 nm for excitation and emission, respectively. Spectraare recorded in single photon counting mode. Changes in lamp intensityare corrected with a quantum counter in the reference channel, measuredin slow mode. Gain is set to ×100 with a high voltage (HV) of 640 V.Integration time is 2 seconds. For excitation spectra measurements, theemission wavelength is set to 397 nm-and the sample is excited from 200to 400 nm. For recording emission spectra, the sample is excited at 342.nm and the emission is recorded from 350 to 540 nm. The data areaveraged over two scans.

Quantum Yield Determination of 4e [Demas & Cosby, 1971; Chen et al.,1972]:

The quantum yield of 4e is determined using chininsulfate as a standard[Melhuish, 1961].

The point of intersection between the UV spectra of 4e and chininsulfate(CS) is determined by measurements of a solution of the two substanceson a Cary 1E spectrophotometer. Concentrations were 25 μM in THF for 4eand 50 mM in 1N H₂SO₄ for CS. Spectra are recorded from 260 nm to 380 nmusing a quartz cuvette with an optical pathlength of 10 mm, and blankcorrected by subtracting the solvent absorption. The point ofintersection of the two resulting UV spectra is determined to be at348.3 nm, corresponding to 352.8 nm on the SLM-8000 C fluorometer due tomonochromator shifts. The fluorescence emission spectra of 4e and CS arerecorded using the same solutions as for the UV-measurements in singlephoton counting mode. Lamp intensity changes are corrected with aquantum counter in the reference channel, measured in slow mode. Gain isset at ×100, high voltage applied was 830 V. Integration time was 0.5seconds.

Spectral bandwidths are set at 1 nm for excitation, 2 nm and 4 nm foremission. For the recording of the emission spectra, excitation is setto 352.8 and emission is monitored from 360 to 550 nm for IAE and 360 to650 nm for CS.

Calculation of R₀ (Förster Distance) Between 4e and N-acetyl-tryptophanamide (NATA):

To calculate R₀ between 4e (acceptor) and NATA (donor), the emissionspectrum of NATA and the absorption spectrum of 4e are used to determinethe region of overlap between 300 nm and 380 nm. The emission spectrumof NATA is measured at a concentration of 15 μM in THF, using a 750 μlquartz cuvette with an optical pathlength of 10 mm. Measuring mode issingle photon counting. Changes in lamp intensity are corrected using aquantum counter in the reference channel. The gain is set at ×100, thehigh voltage applied was 640 V and the integration time is 2 seconds.The fluorescence emission is corrected for photometric accuracy of thesystem by multiplication with-correction factors obtained by standardlamp calibration. Slit widths are set at 1 nm and 4 nm for excitationand emission, respectively. The sample is excited at 293 nm and emissionwas monitored from 300 to 480 nm.

R₀ is calculated using the formulaR ₀=(9.97×10³)(Jκ ²η⁻⁴Θ_(NATA))^(1/6) Åwhere κ² is an orientation factor of the relative orientation in spaceof the transition dipoles of the donor and acceptor chromophores. Forthe donor-acceptor pairs that have isotropic rotation on the time scaleof the fluorescence lifetime, a value of ⅔ can be used [Chen, 1972].

η is the refractive index of the solvent, in this case THF with a valueof 1.4064. Θ_(NATA) is the quantum yield of the donor, which is takenfrom the literature as 0.12-0.14 [Szabo & Rainer, 1980; Petrich et.al.,1983].

J is the spectral overlap integral which can be approximated fromJ=SF _(NATA)(λ)λ⁴Δλ   (2)where F_(NATA) is the corrected fluorescence emission of the donor, thesum normalized to unity, ε_(4e) is the extinction coefficient of theacceptor at wavelength λ, and DS is the incremental detection range.FRET Measurements:

Spectral bandwidths are set to 8 and 16 nm for excitation and emission,respectively. Measurements are performed at 25° C. in a 750 μL quartzcuvette (10 mm optical pathlength) without stirring after initialmixing. A concentration of 50 nM of 9 or 11 is used for complexationwith 1 μM of (fluorescent) avidin. With a dissociation constant in therange of 10⁻¹⁵ M, as usual for avidin biotin interactions, completesaturation of the labelled biotin platform is achieved. All titrationsare performed in magic angle setting, with the emission wavelength setto 500 nm in experiments where the indazole is the acceptor, and with anexcitation spectrum recorded from 250 nm to 360 nm. In experiments whereindazole is used as donor and Lucifer Yellow or BODIPY FL is theacceptor, the excitation wavelength is set to 338 nm or 325 nm. Thefluorescence emission is collected between 360 nm and 700 nm. Thespectra are corrected for Raman scattering from the PBS solution, fordilution, and for inner filter effects due to protein additions. Thesignal is adjusted for lamp intensity changes by ratio mode detectionwith a Rhodamine B quantum counter in the reference channel. A WG 360emission cutoff filter is used for straylight reduction. Data arecollected in 2 nm increments with 1 second integration time.

Fluorescence Anisotropy Measurements:

Fluorescence anisotropy is calculated according to [Lakowicz, 1983].

Spectral bandwidths are set to 4 and 8 nm for excitation and emission,respectively, with 5 seconds integration time. For calculation offluorescence anisotropy ten data points are averaged.

Picosecond Time-Resolved Fluorescence Spectroscopy:

Picosecond time-resolved fluorescence measurements are recordedaccording to the single photon counting technique [Lakowicz, 1983 p52-93, 112-153, 156-185]. An Ar⁺-pumped Ti:Sapphire laser (model MIRA900, Coherent, Santa Clara, USA) provided under mode-locked conditionsultrashort pulses (FWHM=150 fs) with a frequency of 75 MHz. To reducethe repetition rate to 4.7 MHz, a pulse picker (model 9200, Coherent,Santa Clara, USA) is inserted into the beam. The beam is then split totrigger a fast photo diode on the one hand, which yields afterdiscrimination (Ortec pico-timing discriminator 9307, Oak Ridge, USA)the stop signal for the SPC measurements (reverse mode), and to excitethe sample on the other hand. The excitation wavelength of 360 nm isobtained by first manually tuning the MIRA 900 to 720 nm and then doublethe frequency of the beam with the ultrafast harmonic generation system5-050 (Inrad, Northvale, USA). The excitation power ranged between0.1-0.3 mW, the full width at half maximum of the instrument responsefunction being 70-80 ps. Fluorescence photons are collected in a Spex1681 single grating monochromator (Spex, Edison, USA) and detected withthe Hamamatsu R3809U microchannel plate (Hamamatsu, Shimokanzo, Japan).The so obtained start signal is preamplified (preamplifier 9306, Ortec,Oak Ridge, USA) and discriminated (discriminator 9307, Ortec, Oak Ridge,USA) before starting the voltage ramp in the time-to-amplitudeconverter, which is stopped in the reverse SPC mode by the next signalof the photodiode. The voltage values which correspond to the timepassed between excitation and emission of a fluorescence photon in asingle experiment are stored in the Spectrum Master 921 (Ortec, OakRidge, USA) which is connected to the multichannel emulation programMaestro (Ortec, Oak Ridge, USA). SPC raw data are iterativelyreconvoluted with the FLA900 program from Edinburgh Instruments(Edinburgh, UK) according to the multiexponential modelF(t)=A+εb _(i)exp(−(t+δt)/ρ_(i))where A represents the background noise of the experiment, b_(i)represent the pre-exponential terms of the according fluorescencelifetimes ρ_(i), and δt is an optional temporal shift term to compensatefor shifts of the instrument response. Relative amplitudes B_(i) areobtained by computingB _(i)=[(b _(i)ρ_(i))/ε(b _(i)ρ_(i))]×100(%).

The time resolved decay of 4e is determined at a concentration of 25 μMin THF. Excitation is from 350 nm 380 nm, fluorescence emission isdetected at 400 nm with slit widths set to 0.5 nm. Mean width at-halfmaximum of the laser pulse is 130 fsec, of the excitation pulse 65 psec.15 000 counts are collected in the maximum.

EXAMPLE 1A Detailed Spectroscopic Characterization of Selected Example4-[3-(4-allyloxycarbonyl-phenyl)-1H-indazol-1-yl]-benzoic acid (4e)

The AIDA derivative 4e shows absorption maximum at 342 nm and a maximalemission intensity at 403 nm. The compound has a large Stoke shift ofabout 60 nm and is energetically ideally located between emission oftryptophans in most macromolecular environments and many fluorophoreswhich are commercially available and used as tracers in biochemistry(FIG. 1-2). The almost perfect overlap between the absorption andfluorescence excitation spectra indicates low probability ofphotochemical and photophysical effects in the excited state.

The absorption coefficient, ε, at 337 nm is 29926 M⁻¹ cm⁻¹ which isabout 50% of the value known from fluorescein or rhodamine derivatives,but high enough to serve as acceptor in FRET measurements in thenanomolar concentration range. The quantum yield of 4e, Q_(4e) is 0.591.In comparison to tryptophan (≈0.1) or fluorescein (in organicsolvents≈0.9, linked to proteins or RNA≈0.2-0.4), for a small compound,like 4e, this is a surprisingly high quantum yield suggesting goodfluorescence donor properties.

Förster distance to NATA:

R₀ is defined as the distance at which the transfer rate (k_(T)) isequal to the decay rate of the donor in the absence of acceptor. At thisdistance one-half of the donor molecules decay by energy transfer andone-half by the usual radiative and non-radiative processes. ThereforeR₀ can be used the check whether indazole derived platform molecules canbe used as donor or acceptor in energy transfer experiments in a commonsituation of distances in protein ligand interactions.

The R₀ value of the dye pair 4e and NATA is between 25.2-26.3 Å Withaverage linker distances between 4 and 10 Å separating the fluorescentindazole tracer from the potential inhibitor, it is very likely that atryptophan in a substrate binding site is within another 15-20 Å. On theother hand, labeling frequency for external tracers can be adjusted toobtain at least one molecule of the label within 20 Å of the ligandbinding site.

AIDA derivative 4e showed a mono-exponential decay with a fluorescencelifetime λ₁ of 1.055±0.117 nsec with a relative amplitude of 99.73%.

Conclusion: 4e is photophysically and photochemically stable withpromising spectroscopic characteristics as fluorescent linker moleculefor detection of ligand binding to target proteins.

EXAMPLE 2A Detection of Binding of AIDA-Biotin Conjugatetriethylammonium;(2-4-[3-(4-allyloxycarbonylphenyl)-1H-indazol-1-yl]-benzoylamino}-6-((+)-biotinoylamino)-hexanoate(11) to avidin

With a K_(d) of 1.3×10⁻¹⁵ M at pH 5 the avidin-biotin complex representsone of the most stable biomolecular interactions known. Both componentsare extensively characterized. To investigate the properties of AIDAdyes as reporter or detector of interactions with a protein bindingsite, biotin therefore represents an ideal ligand to mimic a potentialinhibitor. The binding site for biotin in avidin comprises 4tryptophans. Two, tryptophan 70 and 97 are within 5 Å from the biotin inthe binding pocket. Tryptophan 10 is 12 Å in distance from biotin,tryptophan 110 in 20 Å, respectively. 11 was evaluated for its spectralproperties in PBS buffer and for the overlap with the tryptophan,Lucifer Yellow and BODIPY-FL fluorescence in avidin (FIG. 2 b, 2 e, 2h).

FIG. 2 b: Excitation and emission spectra of 11 and avidin showing theoverlap between tryptophan emission and excitation spectra of 11.

AIDA derivative 11 has very broad fluorescence emission bandwith withabout 50% of emission intensity at 500 nm. The spectral overlap betweenthe tryptophan emission in avidin and the absorption of 11 is almostperfect.

FIG. 2 e: Spectral overlap of 11 with Lucifer Yellow labelled avidin.

Lucifer Yellow was chosen as possible acceptor for the indazoles becauseof its absorption between 400 and 500 nm. The overlap with the emissionspectrum of 11 in water is almost quantitative.

BODIPY FL absorbs between 480 and 500 nm with an approximately tenfoldhigher extinction coefficient than Lucifer Yellow. It can be used ascheck for the relative importance of the extent of the overlap integralin relation to ε.

Conclusion: The spectral overlap integral between donor (tryptophan) andacceptor (Lucifer Yellow, BODIPY FL) labels in avidin and thefluorescent AIDA molecule show the ideal energetic position of theindazole for interaction studies. It therefore remains a sole questionof distance and non-radiative depopulation terms, whether efficientenergy transfer will take place or not.

AIDA Conjugate 11—Avidin Complexes:

(a) Fluorescence anisotropy of 11 as signal for the biotin binding toavidin in the model system.

AIDA conjugate 11 at a concentration of 50 nM, had a basic fluorescenceanisotropy of 0.034 when monitored at the excitation wavelength of 338nm and the emission maximum of 438 nm. This value is indicative for asmall monomeric molecule rotating free in solution. When bound to avidinadded in excess of 1 μM, the anisotropy value was 0.151 (+444%)representing. the fixation of the indazole moiety attached to biotin ator near the binding site on avidin. When 1 μM of the BODIPY-FL labelledavidin was used as substrate the r-value was 0.100. This reduced changein anisotropy probably reflects the lower affinity of the BODIPY-FLlabelled avidin as compared to the unlabelled material.

(b) Resonance Energy Transfer experiments with 11 as Donor or Acceptor.

FIG. 3 a shows the fluorescence excitation spectra of 50 nM of 11 and 1μM of unlabelled avidin. An emission wavelength at 480 nm was chosenbecause the tryptophan fluorescence of avidin had almost reached thebaseline level at this emission energy, resulting in only minor avidinfluorescence contribution between 250 and 370 nm. The differencespectrum: [I(11_((free)))+I(avidin_((free)))]−I(11-avidin_(complex)),FIG. 3 a curve 5, indicates that the fluorescence of 11 is quenched byabout 25.5% by complexation in the avidin binding site. This quenchingeffect can be attributed to all radiative and non-radiative processesoccurring to 11 in the vicinity of the tryptophans in the binding cleft.Corresponding to the ratio of the negative and positive areas under thefluorescence difference curve (black), 62% of this total quenchingeffect is fluorescence resonance energy transfer. From 250 to 305 nm theenhancement of the tryptophan and tyrosine fluorescence intensity is 72%based on the sum of 11 and avidin.

Conclusion: When 11 is used as acceptor and protein fluorescence inavidin as donor, both an energy transfer to, and a quenching effect on11 can be used as a signal for the complex formation.

FIG. 3 c shows the fluorescence emission spectra of 50 nM of 11, 1 μMavidin-BODIPY-FL, the complex- and the relevant difference spectra. Inthese experiments 11 was used as donor and BODIPY-FL as acceptor. At anexcitation wavelength of 338 nm the emission of 11 between 360 nm and516 nm is strongly quenched by approximately 80% in the complexed form.The standard definition for energy transfer efficiency,E=1−F_(da)/F_(d), would result in E≅0.8. For intermolecular FRETmeasurements or for intramolecular FRET determinations where a secondcomponent induces potential conformational changes in the binding sitesthis “classical” definition for E is not valid. The approximately 80%reduction in signal intensity of 11 can be caused either by FRET or fromlocal quenching of the fluorescence of 11 mainly by ground stateinteractions. However, a small enhancement in BODIPY-FL -fluorescenceintensity between 500 and 600 nm, where energy transfer becomes visible,suggests ground state complexation to be the main reason for thequenching effect. However, it can not be excluded, that the BODIPY-FLlabel looses its excitation energy by non-radiative decay processes,although most of the emission energy of 11 was absorbed by BODIPY-FL.The extinction coefficient of approximately 90 000 M⁻¹ cm⁻¹, determinesthat the quenching effect of almost 80% on 11 between 360 nm and 516 nmis an extremely efficient signal for biotin interaction with avidinmonitored by 11 at wavelengths between 440 nm and 500 nm. Due to thebroad fluorescence emission of 11 in water the detection wavelength canbe shifted up to 500 nm which is at lower energy than most backgroundfluorescence signals from small chemical compounds.

Conclusion:

1. Fluorescence anisotropy/polarization allows to monitor complexformation with high sensitivity. Based on the photophysical relationbetween fluorescence anisotropy and rotational and translationaldiffusion it, can be concluded that 11 and related AIDA dye derivativesare suitable tracers in single-molecule specroscopy as well.

2. With avidin-biotin as model-system a tryptophan to 11 energy transferof 70% relative to free avidin plus 11 was detected at 480 nm asefficient signal for complex formation.

3. 70-80% reduction of fluorescence intensity of 11 which can compriseFRET, ground-state quenching of indazole and excited state quenching ofthe avidin label BODIPY-FL provides a third possibility for monitoringthe protein-ligand complex between 440 and 500 nm.

EXAMPLE 3A Detection of Binding of AIDA-Biotin Conjugate4-[1-[4-carbamoylphenyl]-1H-indazol-3yl]-benzoic acid3-(biotinoylamino]-propyl amide (8) to avidin

(a) Excitation and emission spectra of 8 and avidin showing the overlapbetween tryptophan emission and excitation spectra of 8 are presented inFIG. 2 c. Wheras 11 has a very broad fluorescence emission band withabout 50% of emission intensity at 500 nm (FIG. 2 b) the fluorescenceemission spectrum of 8 with the biotin linked to the indazole position 3lacks the broad transition of 11 with a maximum at 450 nm, resulting ina much narrower spectrum. FIG. 2 f and FIG. 2 i show the spectraloverlap integral between donor (the AIDA fluorescent molecule) andacceptor, Lucifer Yellow and BODIPY FL labels, respectively, in avidin.The different overlap integral for fluorescence emission of 11 and 8,respectively, and BODIPY absorption allows a qualitative estimation ofthe relative oriantation factor involved in the energy transfer process.It therefore remains a sole question of distance and non-radiativedepopulation terms, whether efficient energy transfer will take place ornot.

(b) Fluorescence Resonance Energy Transfer experiments with 11 as donoror acceptor.

AIDA Conjugate 8 as Acceptor: (FIG. 3 b)

The difference spectra [I(⁸_((free)))+I(avidin_((free)))]−I(8-avidin_(complex)) (FIG. 3 b, curve5). Linking the biotin to position 3 in the indazole molecule in 8 (fromposition 1 in 11) not only changes the fluorescence spectra, but alsothe tryptophan to indazole FRET characteristics: indicated by 26%increase in indazole fluorescence emission (instead of quenching) anenhanced energy transfer to tryptophan (120%) is superimposed by astrong increase in indazole quantum yield in the avidin environment.

AIDA Congugate 8 as Donor: (FIG. 3 d)

Although the overlap integral between the indazole emission and theBODIPY-FL absorption spectrum is smaller for 8 than for 11, moresensitization of acceptor fluorescence occurs in the 8-avidin-BODIPYcomplex resulting in 48% fluoresence energy transfer and 93.5% indazoledonor quenching. This effect can be caused by a higher donor quantumyield or a prefered relative orientation of the donor emission andacceptor absorption dipoles in 8.

Conclusions:

With both molecular geometries, the indazole tracer linked via position1 (11) and position 3 (8), binding of BODIPY-FL labelled avidin causes a70-90% quenching of the indazole emission. This reduction in signalintensity can be caused by ground state quenching of the indazole orFRET to the BODIPY labels in avidin with concurrent non-radiativedepopulation of the excited state. Besides of the donor quenching signalthe acceptor sensitization will provide a selective interaction signalof AIDA conjugates with target macromolecules labelled withconventiuonal dyes fluorescing in >500 nm with FRET efficiencis of 40%and higher.

c. Binding of AIDA conjugate 8 to avidin-BODIPY detected by rotationaldiffusion and energy transfer: Energy transfer between the indazolemoiety in 8 and the 3.3 BODIPY-FL lables on avidin by time-resolvedfluorescence spectroscopy.

AIDA conjugate 8 exhibits fast fluorescence depolarisation in aqueoussolution (PBS/5% DMSO), common to chromophores attached to a smallmolecule. Binding of avidin (unlabelled) leads to a slowerdepolarisation of 8 due to the decreased rotational diffusion of thecomplex. Binding of avidin labelled with BODIPY-FL to 8 creates afurther depolarisation mode for the indazole fluorescence: Fluorescenceresonance energy transfer between the indazole moiety of 8 and theBODIPY-FL labels. Therefore, although 8 binds to avidin via biotin, therotational diffusion of the 8 bound to avidin-BODIPY monitored at theindazole emission wavelength is quickly depolarised, like in the unboundform. When the 8-avidin BODIPY-FL complex is monitored at 550 nm (BODIPYemission wavelength), the rotational diffussion measured by thefluorescence anisotropy decay is as just a little bit slower than in theuncomplexed avidin-BODIPY construct.

Conclusion: The interaction between fluorescenctly labelled targetmacromolecules and AIDA conjugates can also be detected in thetime-resolved set-up for fluorescence measurements by either monitoringchanges in fluorescence lifetimes, rotational correlation times, orrotational or translational diffusion times in single moleculespectroscopy.

Part B: Chemical

General

¹H-NMR spectra are recorded with a Bruker WC-250 or AMX-500spectrometer; chemical shifts are given in ppm (ä) relative to Me₄Si asinternal standard. J values are given in hertz and are based upon firstorder spectral interpretation. Elemental analyses are performed by theAnalytical Department, Novartis Basle, Switzerland and are within ±0.4%of the theoretical value unless otherwise stated. ESI-MS spectra areobtained on a Finnigan-SSQ 7000, and CI-MS spectra on a Finnigan-TSQ700. Melting points are determined with a Thermovar microscope(Reichert-Jung) and are uncorrected. Analytical thin layerchromatography (TLC): Merck precoated silica gel 60 F254 plates;detection: (a) λ=254 nm, (b) λ=366 nm, (c) staining with MOSTAIN (5%(NH₄)₆Mo₇O₂₄×4 H₂O, 0.1% Ce(SO₄)₂ in 10% H₂SO₄) or (d) staining withNinhydrin (sat. soin. in ethanol) and subsequent heating on a hot plate.Medium pressure chromatography (MPLC): silica gel Merck 60 (40-6.3 μm).Reagents and solvents are purchased in analytical or the best availablecommercial quality and used without further purification unlessotherwise stated. Evaporations are carried out in vacuo with a rotaryevaporator; drying at high vacuum (HV) is performed under an oil pumpvacuum better than 0.1 mbar.

Abbreviations

BOP (Benzotriazol-1-yloxy)-tris-(dimethylamino)-phosphoniumhexafluorophosphate (Castro's Reagent)); CI (Chemical Ionization); dec.(decomposition); DCM (DichloromethaneDIC (Diisopropylcarbodiimide); DIEA(Diisopropyl ethyl amine (Hunig's base)); DMA (N,N-Dimethylacetamide);DMF (Dimethylformamide); DMEU (N,N′-Dimethylethylenurea); DMPPU(N,N′-Dimethylpropylenurea); DMSO (Dimethylsulfoxide); ESI (ElectrosprayIonization); Fmoc (9-Fluorenylmethoxycarbonyl); HOBt(1-Hydroxybenzotriazole); HV (High vacuum); m.p. (Melting point); MS(Mass spectrum); n.d. (not determined); RT (Room temperature); TEA(Triethylamine); TEAA (Triethylammoniumacetate); TFA (Trifluoro aceticacid);THF (Tetrahydrofuran); TSTU(O-(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate).

Synthetic Procedures EXAMPLE 1B (a) General Procedures for the Synthesisof Functionalized 1,3-Diaryl-1H-indazoles (4a-j) as illustrated inScheme 1:

Scheme I

1-4 a b c d e f g h i j R1 H H H H H H Cl MeO H CH₂ R2 H CO₂H CONH₂CO₂CH₃ CO₂allyl NO₂ Cl MeO CO₂H NO₂ R3 CO₂H CO₂H CO₂H CO₂H CO₂H CO₂HCO₂H CO₂H NO₂ CO₂HConditions:(a) 4-Hydrazino-benzoic acid or 4-nitro-phenylhydrazine, MeOH, reflux,50 h.(b) lead-tetraacetate, DCM, RT 30-60 min.(c) DCM, boron-trifluoride etherate, 0° C., 20 min, RT, 30 min.

Synthesis of Arylhydrazones (2a-j) from Diarylketones (1a-j) asillustrated in Scheme 1. A suspension of the diarylketone (1a-j; 10mmol) and the arylhydrazine (11 mmol) in methanol (50 mL) is refluxedfor 50 h. Upon heating a clear solution is obtained. During the courseof the reaction a crystalline precipitate is formed. After cooling in anice bath the crystalline product (2a-j) is collected by filtration,washed twice with cold methanol and several times with diethylether. Thecrystals of 2a-j are kept overnight in a drying oven at 70° C.Unsymmetrically substituted diarylketones (1b-f, 1i-j) yield mixtures ofE/Z-isomers (2b-f; 2i-j), which are introduced in the next reaction stepwithout separation. Yields and analytical data of 2a-j: 2 a b c d e f gh i j Yield 77 80 79 83 92 87 66 78 99 99 [%] m.p. 249- 40:60* 46:54*32:68* 41:59* 33:67* 271- 255- 50:50* 34:66* [° C.] 251 273 257 [MH]⁺317 315 360 375 401 360 384 377 362 376 [M-H]⁻ −CO₂H*ratio of E/Z-isomers (not assigned); ¹H/¹³C-NMR spectra: in agreementwith the structures of 2a-j.

Synthesis of Acetic Acid 1,1-diaryl-1-arylazomethyl Esters (3a-j) asillustrated in Scheme 1. An arylhydrazone of 2a-j (10 mmol) is dissolvedin dichloromethane (50 mL; complete solution can be obtained by additionof 10% acetic acid in case of low solubility of the starting material).A solution of lead tetraacetate (10 mmol) in dichloromethane (10 mL) isadded at room temperature. The homogeneous reaction mixture is stirredfor 30-60 min. The completion of the reaction is monitored by thin layerchromatography. A suspension of sodium oxalate (1 g in water) is addedto the orange-yellow solution. After 15 min of vigorously stirring theentire mixture is filtered. The aqueous layer is discarded. The organiclayer is extensively washed with water and evaporated under reducedpressure. The remaining orange oil is dissolved in ethyl acetate. Theorganic phase is washed again with several portions of water, dried overMgSO₄ and evaporated to dryness under reduced pressure. The remainingorange foam of 3a-j is further dried at room temperature in vacuoovernight. 3a-j are homogeneous as is indicated by thin layerchromatography and are introduced in the next reaction step withoutfurther purification. Yields and analytical data of 3a-j: 3 a b c d e fg h i j Yield-% 88 93 93 97 99 99 99 99 90 99 [MH]⁺ 271 359 358 373 399418 441 375 360 374 [M-H]⁻ (−OAc) (−OAc) (−OAc) (−OAc) (−OAc) (−OAc)¹H/¹³C-NMR spectra: in agreement with the structures of 3a-j.

Synthesis of 1,3-diaryl-1H-indazoles (4a-j) as illustrated in Scheme 1.An acetic acid 1,1-diaryl-1-arylazomethyl ester of 3a-j (10 mmol) isdissolved in dichloromethane (50 mL). The solution is cooled to 0° C. inan ice bath. Boron trifluoride etherate (13 mmol) is added understirring within 5 min. The addition of boron trifluoride etherateresults in instantaneous formation of a precipitate. The reactionmixture is stirred at 0° C. for 20 min and at room temperature foradditional 30 min. The crystalline precipitate is collected by vacuumfiltration. The crystals are washed twice with cold methanol and threetimes with diethylether. The 1,3-diaryl-1H-indazoles 4a-j are driedunder reduced pressure in a drying oven at 70° C. Thin layerchromatography indicates homogeneity of 4a-j (recrystallization can beachieved from hot dimethylformamide, either pure or upon addition ofmethanol). Yields and analytical data of 4a-j: 4 a b c d e f g h i jYield 72 92 91 93 99 64 78 33 47 82 [%] m.p. 266- 320 322- 313- 238-306- 341- 243- 273- 355- [° C.] 268 (dec.) 323 314.5 240 307 342 246 275360 [MH]⁺ 315 359 358 373 399 359 382 375 360 n.d. [M-H]⁻

(b) Synthesis of 1,3-Diaryl-5-nitro-1H-indazoles (4k, 4l)

4-[3-(4-carboxy-phenyl)-5-nitro-1H-indazol-1-yl]-benzoic Acid (4k).Nitric acid (65%, 4 mL) is cooled to 0° C. in an ice bath and sulfuricacid (95%, 4 mL) is added. 1,3-Bis-(4-carboxy phenyl)-1H-indazole (4b)(300 mg, 0.835 mmol) is added at 0° C. under stirring. Cooling andstirring is continued for 10 min. The mixture is poured into ice-water(150 mL) and pH is adjusted to 4-5 by addition of 2N-NaOH. The crystalsare collected by filtration, washed with brine until the filtrate wasclose to neutral. The crystals are dried for 2 h at 90° C. andrecrystallized from dimethylformamide/methanol. The product is collectedby filtration and dried at 120° C. under reduced pressure yielding 153mg (45 %) of 4k: m.p. 348-350° C.

4-[3-(4-carbamoyl-phenyl)-5-nitro-1H-indazol-1-yl]-benzoic Acid (4l).Nitric acid (65%, 4 mL) is-cooled to 0° C. in an ice bath and sulfuricacid (95%, 4 mL) is added.4-[3-(4-carbamoyl-phenyl)-1H-indazol-1-yl]-benzoic acid (4c) (300 mg,0.84 mmol) is added at 0° C. under stirring. Cooling and stirring iscontinued for 15 min. The mixture is poured into ice-water (150 mL). Theprecipitate is collected by centrifugation, washed with brine until thefiltrate was close to neutral. The crystals are dried for 30 min at 120°C. and recrystallized from dimethylformamide/methanol. The product iscollected by filtration and dried at 120° C. under reduced pressureyielding 211 mg (62%) of 41: m.p. 320-322° C.

NMR Spectral Data of 4a-l:

4a: ¹H-NMR (DMSO-d₆): 7.36-7.47 (m, 1H); 7.47-7.50 (m, 1H); 7.54-7.59(m, 3H); 7.99-8.02 (m, 3H); 8.03-8.06 (m, 2H); 8.14-8.18 (m, 3H).¹³C-NMR (DMSO-d₆): 111.4; 121.5; 121.7; 123.0; 123.1; 127.6; 128.2;128.4; 128.9; 129.2; 131.1; 132.3; 139.7; 143.1; 146.2; 166.8 (COOH).

4b: ¹H-NMR (DMSO-d₆): 7.40 (ddd, 1H); 7.60 (ddd, 1H); 7.95-8.25 (m,12H); 13.0 (broad, 2H). ¹³C-NMR (DMSO-d₆): 112.0; 122.1; 122.3; 123.4;123.9; 127.9; 128.8; 129.2; 130.6; 131.2; 131.6; 136.8; 140.3; 143.3;145.5; 167.3 (COOH); 167.6 (COOH).

4c: ¹H-NMR (DMSO-d₆): 7.42 (ddd, 1H); 7.45 (broad, 1H, CONH); 7.62 (ddd,1H); 8.02-8.06 (m, 3H); 8.08-8.10 (m, 2H); 8.12 (broad, 1H, CONH);8.15-8.19 (m, 4H); 8.22-8.25 (dd, 1H); 12.5 (broad, 1H, COOH). ¹³C-NMR(DMSO-d₆): 111.8; 122.0; 123.2; 123.6; 127.5; 128.7; 128.9; 131.4;134.6; 135.2; 140.1; 143.2; 145.6; 167.1 (CONH₂); 167.9 (COOH).

4d: ¹H-NMR (DMSO-d₆): 3.85 (s, 3H); 7.43 (t, 1H); 7.62 (t, 1H);8.00-8.24 (m, 10H). ¹³C-NMR (DMSO-d₆): 52.3 (OCH₃); 111.5; 121.5; 121.8;122.8; 123.4; 127.5; 128.3; 128.6; 129.4; 129.9; 131.0; 136.7; 139.7;142.7; 144.7; 166.0; 166.7.

4e: ¹H-NMR (DMSO-d₆): 4.84-4.86 (m, 2H); 5.29-5.32 (m; 1H); 5.42-5.46(m, 1H); 6.05-6.12 (m, 1H); 7.41-7.44 (m, 3H); 8.07-8.19 (m; 4H);8.21-8.24 (m, 3H). ¹³C-NMR (DMSO-d₆): 65.4; 111.6; 118.2; 121.7; 121.9;122.9; 123.5; 127.7; 128.4; 128.8; 129.5; 130.1; 131.1; 132.8; 136.9;139.9; 142.9; 144.8; 165.3 (CO—O-allyl); 166.8 (COOH). Microanalysis:(C₂₄H₁₈N₂O₄) calculated, C, 72.35; H, 4.55; N, 7.03; found, C, 72.13; H,4.60; N, 7.02.

4f: ¹H-NMR (DMSO-d₆): 8.6-7.2 (m; aromat. H). ¹³C-NMR (DMSO-d₆): 112.12;121.9; 122.5; 123.2; 124.2; 124.8; 128.7; 128.9; 129.4; 131.5; 140.2;140.4; 143.1; 143.3; 147.6; 167.2 (CO₂H).

4g: ¹H-NMR (DMSO-d₆; 330° K): 8.15 (m, 3H), 8.05 (m, 3H), 7.98 (m, 2H),7.60 (m, 2H), 7.40 (ddd, 1H). ¹³C-NMR (DMSO-d₆, 330° K) 111.8; 122.3;122.7; 123.9; 124.5; 130; 131.4; 131.8; 134.3; 134.6; 141.1; 143.2;145.9; 167.5 (CO₂H).

4h: ¹H-NMR (DMSO-d₆): 3.86 (s, 3H, OCH ₃); 3.92 (s, 3H, OCH ₃); 7.00(dd; 1H); 7.10 (m, 2H); 7.35 (d, 1H); 7.95-8.05 (m, 5H); 8.15-8.22 (m,2H); 12.0 (broad, 1H, COOH). ¹³C-NMR (DMSO-d₆): 55.2 (OCH₃); 55.6(OCH₃); 92.8; 113.9; 114.4; 117.3; 121.3; 122.4; 124.7; 127.9; 128.6;130.9; 140.9; 143.1; 145.8; 159.7; 160.0; 166.7 (COOH).

4i: ¹H-NMR (DMSO-d₆): 7.5 (ddd, 1H); 7.7 (ddd, 1H); 8.1-8.3 (m, 8H);8.45 (m, 2H). ¹³C-NMR (DMSO-d₆): 112.0; 122.2; 122.3 (2x); 123.6; 124.1;125.8 (2×); 127.9 (2×); 129.1; 130.4 (2×); 131.3; 136.2; 140.1; 144.8;145.1; 146.3; 167.4 (C═O).

4j: ¹H-NMR (DMSO-d₆): 2.54 (s, 3H); 7.30 (d, 1H); 7.83 (s, 1H); 8.01 (d,2H); 8.14 (d, 1H); 8.18 (d, 2H); 8.33-8.40 (m, 4H); 12.8 (broad, COOH).

4k: ¹H-NMR (DMSO-d₆): 7.97 (2H); 8.05-8.20 (m, 7H); 8.38 (dd, 1H); 8.93(d, 1H). 11.5 (broad, 2H). ¹³C-NMR (DMSO-d₆): 112.8; 119.2; 122.3;122.8; 123.2; 128.0; 130.1; 130.6; 131.4; 131.7; 135.1; 141.9; 142.1;143.6; 147.4; 166.9 (COOH); 167.3 (COOH).

4l: ¹H-NMR (DMSO-d₆): 7.52 (broad, NH ₂); 8.0 (m, 2H); 8.3-8.05 (m, 7H);8.35 (dd, 1H); 8.94 (d, 1H); 11.2 (broad, COOH). ¹³C-NMR (DMSO-d₆):113.0; 119.6; 122.7; 123.1; 123.5; 128.1; 129.1; 130.3; 131.7; 134.0;135.6; 142.2; 142.5; 143.9; 148.1; 167.2; 168.1.

EXAMPLE 2B (a) Synthesis of4-{3-{4-[(3-Aminopropyl)-aminocarbonyl]-phenyl}-1H-indazol-1yl}-benzoicAcid (5) as Illustrated in Scheme 2.

1,3-diamino-propane (80 mL) is added to 4d (10.0 g, 26.85 mmol). Theheterogeneous reaction mixture is heated to 90° C. under stirring. 4ddissolves once the solvent is at reaction temperature, and the solutionis then left to react for 4 h. The 1,3-diamino-propane is distilled offunder reduced pressure leaving a viscous oil behind. The crude productis diluted with methanol (200 mL) and heated to reflux. Before themethanol reaches boiling temperature, crystalline product starts toprecipitate. The suspension is refluxed for 10 min and then cooled toroom temperature. The crystals are collected by vacuum filtration. Thecolourless crystals are washed with a portion of glacial methanol andthen with several portions of diethyl ether. The product is dried in adrying oven under reduced pressure at 120° C., yielding 9.86 g (89%) of5: mp 285-288° C. (depending on the heating rate; dec.); ¹H-NMR(DMSO-d₆): 1.85-2.05 (m, 2H); 2.90-3.05 (m, 2H); 3.35-3.55 (m, 2H); 7.32(t, 1H); 7.51 (t, 1H); 7.75-8.30 (m, 10H); 9.20 (broad, t, 1H, CONH);¹³C-NMR (DMSO-d₆): 27.5; 36.2; 36.4; 111.2; 121.2; 121.4; 122.4; 122.8;126.9; 127.8; 128.0; 130.4; 134.0; 135.0; 137.0; 139.7; 140.2; 143.9;144.2; 165.8; MS: e/m 415 (MH⁺).

(b) Synthesis of4-{3-{4-[N-3-[(9H-Fluoren-9-yl)-methyloxycarbonylamino]-propyl]-aminocarbonyl}-phenyl-1H-indazol-1-yl}benzoicAcid (6) as illustrated in Scheme 2. Potassium carbonate (2.67 g; 15.45mmol) is added to a suspension of the amino acid 5 (3.20 g; 7.73 mmol)in water (60 mL) and 1,4-dioxane (35 mL). The mixture is stirred for 5min and cooled to 0° C. in an ice bath. Fmoc-chloride (2.20 g; 8.5 mmol)in 1,4-dioxane (35 mL) is added dropwise within 10 min. Cooling isremoved and stirring continued at room temperature for 12 h. A bulkyprecipitate is formed. The aqueous suspension is extracted with diethylether. The pH is adjusted to 1 with dilute HCl and then further dilutedwith brine (100 mL). The precipitate is collected by vacuum filtrationand washed extensively with diethyl ether to achieve lumpy crystals. Theproduct is dried for 24 h at 35° C. in a drying oven under reducedpressure, yielding 4.14 g (84%) of 6: mp 217-219.5° C. (depending on theheating rate, dec.); ¹H-NMR (DMSO-d₆): 1.68-1.77 (m, 2H); 3.05-3.12 (m,2H), 3.25-3.35 (m, 2H); 4.22 (t, 1H, H-9 fluorenyl); 4.33 (d, 2H,OCH₂-9-fluorenyl); 7.27-7.35 (m, 3H), 7.37-7.46 (m, 3H); 7.63 (t, 1H);7.70 (d, 2H, H-fluorenyl); 7.88 (d, 2H, H-fluorenyl); 8.01-8.08 (m, 5H);8.15-8.20 (m, 4H); 8.24 (d, 1H); 8.58 (t, 1H, CONH); ¹³C-NMR (DMSO-d₆):29.6; 37.2; 38.4; 47.0; 65.4; 111.6; 120.2; 120.3; 121.8; 123.0; 123.4;125.2; 125.3; 127.2; 127.3; 127.8; 128.1; 128.4; 128.7; 131.2; 134.7;134.8; 139.9; 140.9; 143.0; 144.1; 145.3; 156.3; 166.0; 166.9; MS: 635(M−H)⁻.

EXAMPLE 3B (a) Synthesis of3′(+)-4-{3-[4-(3′-Biotinoylaminopropyl)-aminocarbonyl]phenyl-1H-indazol-1-yl}-benzoicacid Amide (8) as illustrated in Scheme 3.

Scheme 3:

Rink amide resin (500 mg; 0.28 mmol) is shaken with piperidine (20%) indichloromethane (10 mL) at room temperature for 1 h. The resin is washedwith portions of dichloromethane, methanol and finally dichloromethane,until a sample of the latest filtrate shows no presence of cleavedprotecting group. The resin is shaken in absolution prepared from 6(0.56 mmol), 1-hydroxybenzotriazole (1.68 mmol), N,N′-diisopropylcarbodiimide (2.24 mmol) and dimethylformamide (35 mL) overnight at roomtemperature. The resin is washed thoroughly with several portions ofdimethylformamide, then methanol, and finally dichloromethane. Thewashing continues until the latest filtrate is free of fluorescence.Solvents are evaporated under reduced pressure at room temperature. TheFmoc protecting group of resin bound 6 is removed as described above fordeprotection of the Rink amide resin. To a solution of (+)-biotin (0.84mmol) and 1-hydroxybenzotriazole (2.52 mmol) in dimethylformamide (40mL) is added N,N′-diisopropyl carbodiimide (5.04 mmol). The solution isleft at room temperature for 1 h and then transferred to the resin. Thereaction vessel is gently agitated by circular motion for 24 h. Solventsare removed by vacuum filtration. The resin is washed thoroughly withportions of dimethylformamide, methanol, and finally withdichloromethane. Crude product (8) is cleaved from the solid support bytreatment of the resin with a 1:1 mixture of dichloromethane andtrifluoroacetic acid (15 mL) for 2 min. The solution is collected byvacuum filtration. The resin is further extracted with three portions ofdichloromethane. The solvents are removed under reduced pressure,yielding 8 as viscous oil in high purity as indicated by ¹H-NMR.Triturating with ether induces crystallization. Recrystallization from amixture of dimethylsulfoxide, methanol, and diethyl ether yields 60 mg(33%) of 8: mp 222-225° C.; ¹H-NMR (DMSO-d₆): 1.25-1.66 (m, 6H, biotin);1.67-1.72 (m, 2H, NHCH₂CH ₂CH₂NH); 2.09 (t, 2H, COCH2); 2.56, 2.58,2.79, 2.80, 2.81, 2.82 (2H, CH ₂S, AB-part of ABX); 3.08-3.10 (m, 1H,CHS-biotin); 3.11-3.15 (m, 2H, NCH ₂); 3.30-3.34 (m, 2H, NCH ₂);4.11-4.14 (m, 1H, CH-biotin); 4.28-4.30 (m, 1H, CH-biotin, X-part ofABX); 6.34 (s, broad, 1H, NHCONH); 6.42 (s, broad, 1H, NHCONH); 7.44(ddd, 1H); 7.47 (s, broad, 1H of CONH ₂); 7.61 (ddd, 1H); 7.84 (t, 1H,NHCO); 7.97-8.06 (m, 5H); 8.12 (s, broad, 1H of CONH ₂); 8.13-8.26 (m,5H); 8.59 (t, 1H, NHCO). MS: e/m 640 (MH)⁺. ¹³C-NMR (DMSO-d₆): 25.5;28.2; 28.4; 29.5; 35.5; 36.5; 37.3; 55.6; 56.2; 59.4; 61.2; 111.5;121.9; 122.8; 123.2; 127.3; 128.1; 129.3; 132.4; 134.6; 135.0; 139.9;141.7; 145.0; 162.9; 165.9; 167.3; 172.3. Microanalysis:(C₃₄H₃₇N₇O₄S₁×H₂O) calculated, C, 62.08; H, 5.98; N, 14.91; S, 4.87;found C, 62.19; H, 5.82; N, 14.78; S, 4.67.

(b) Synthesis of4-[3-(4-Methoxycarbonyl-phenyl)-1H-indazol-1-yl]-benzoic Acid Amide (7a)as illustrated in Scheme 3. Rink amide resin (1.786 g; 1.00 mmol) isdeprotected as described previously. The deprotected resin is shaken ina solution prepared from 4d (745 mg; 2.00 mmol), 1-hydroxybenzotriazole(810 mg, 6.00 mmol), N,N′-diisopropyl carbodiimide (1.010 g, 8.00 mmol)and dimethylformamide (100 mL) overnight at room temperature. The resinis washed thoroughly with several portions of dimethylformamide, thenmethanol, and finally dichloromethane. The washing continues until thelatest filtrate is free of fluorescence. Solvents are evaporated underreduced pressure at room temperature. An aliquot of product (7a) iscleaved from the solid support (200 mg) by treatment of the resin with a2:1 mixture of dichloromethane and trifluorbacetic acid (10 mL) for 10min. The solution is collected by vacuum filtration. The resin isfurther extracted with three portions of dichloromethane, followed bytwo portions of dimethylformamide. Removal of the solvents under reducedpressure and drying overnight in a desiccator provides 34.0 mg (98%) of7a: mp 267-269° C.; homogeneous by thin layer chromatography:R_(f)=0.68; dichloromethane: methanol=9:1); ¹H-NMR (DMSO-d₆): 3.92 (s,3H, OCH ₃); 7.43 (ddd, 1H); 7.61 (ddd, 1H); 7.95-8.00 (m, 3H); 8.13-8.16(m, 4H); 8.23-8.25 (m, 3H); CONH ₂ broad (not assignable); ¹³C-NMR(DMSO-d₆): 51.9 (COOCH₃); 121.2; 121.6; 122.6; 123.0; 127.3; 127.9;129.0; 129.4; 129.7; 132.5; 136.8; 139.9; 141.4; 144.4; 165.9 (CONH₂);167.03 (COOCH₃).

(c) Synthesis of4-{3-{4-{3-[(9H-Fluoren-9-yl)-methyloxycarbonylamino]-propyl}-aminocarbonyl}-phenyl-1H-indazol-1-yl}benzoicAcid Amide (7b) as illustrated in Scheme 3. Rink amide resin (1.00 g;0.56 mmol) is deprotected as described previously. The deprotected resinis shaken in a solution prepared from 6 (1.12 mmol),1-hydroxybenzotriazole (3.36 mmol), N,N′-diisopropyl carbodiimide (4.48mmol) and dimethylformamide (50 mL) overnight at room temperature. Theresin is washed thoroughly with several portions of dimethylformamide,then methanol, and finally dichloromethane. The washing continues untilthe latest filtrate is free of fluorescence. Solvents are evaporatedunder reduced pressure at room temperature. An aliquot of product (7b)is cleaved from the solid support (51 mg) by treatment of the resin witha 2:1 mixture of dichloromethane and trifluoroacetic acid (3 mL) for 10min. The solution is collected by vacuum filtration. The resin isfurther extracted with three portions of dichloromethane, followed bytwo portions of dimethylformamide. Removal of the solvents under reducedpressure provides 40.5 mg (88%) of 7b as an amorphous solid in highpurity as indicated by ¹H-NMR and thin layer chromatography (R_(f)=0.64;dichloromethane: methanol=9.5 : 0.5;+trace of acetic acid); ¹H-NMR(DMSO-d₆): 1.71 (m, 2H, CONHCH₂CH ₂CH₂NHCOO); 3.09 (m, 2H, NHCH₂CH₂CH₂NHCOO); 3.32 (m, 2H, CONHCH ₂CH₂CH₂NHCOO); 4.22 (distort. t, 1H, H-9fluorenyl); 4.32 (distort. d, 2H, COOCH ₂-(9-fluorenyl); 7.30-7.35 (m,2H, fluorenyl; 1H, —CONHCH₂CH₂CH₂NHCOO)); 7.40-7.44 (m, 2H, fluorenyl;1H, indazole); 7.47 (s, broad, 1H of —CONH ₂); 7.60-7.63 (ddd, 1H,indazole); 7.70 (d, 2H, fluorenyl); 7.88 (d, 2H, fluorenyl); 7.97-8.06(m, 5H, indazole); 8.12 (s, broad, 1H of —CONH ₂); 8.13-8.25 (m, 3H,indazole); 8.58 (t, 1H, CONHCH₂CH₂CH₂NHCOO).

EXAMPLE 4B (a) Synthesis of4-[3-(4-Allyloxycarbonyl-phenyl)-indazol-1-yl]-benzoic Acid2,5-Dioxo-pyrrolidin-1-yl Ester (10) as illustrated in Scheme 4.

To a stirred solution of 4e (355 mg, 0.891 mmol) andN-hydroxy-succinimide (106 mg, 0.891 mmol) in dioxane (20 mL),diisopropylcarbodiimide (0.141 mL, 0.891 mmol) is slowly added via asyringe under an argon atmosphere. The mixture is stirred for 72 h atroom temperature and subsequently evaporated to dryness. The residue isredissolved in ethyl acetate (60 mL) and washed consecutively withNH₄Cl_(sat.aq)., NaCl_(sat.aq)., and water (10 mL each). The collectedaqueous phases are extracted once with ethyl acetate (10 mL). Pooledorganic phases are dried over MgSO₄ and evaporated to dryness. Theresulting yellowish, amorphous solid is purified twice by MPLC (eluentof 1st chromatography: cyclohexane: ethyl acetate=2:1; eluent of 2ndchromatography:toluene:ethyl acetate=12:1) to give 370 mg (83%) of 10:mp 178° C.; homogeneous by thin layer chromatography: R_(f)=0.20(cyclohexane:ethyl acetate=2:1). ¹H-NMR (DMSO-d₆): 2.93 (s, 4H);4.85-4.88 (m; 2H); 5.30-5.33 (m, 1H); 5.42-5.47 (m, 1H); 6.05-6.13 (m,1H); 7.45-7.49 (t, 1H); 7.64-7.68 (t, 1H); 8.11-8.32 (m, 10H). MS: m/e496 (MH⁺). Microanalysis: (C₂₈H₂₁N₃O₆) calculated, C, 67.87; H, 4.27; N,8.48; found, C, 67.99; H, 4.34; N, 8.70.

(b)(5S)-4-{1-[4-(5-Carboxy-5-(biotinoylamino)-pentylcarbamoyl)-phenyl]-1H-indazol-3-yl}-benzoicAcid Allyl Ester Triethylammonium salt (11) as illustrated in-Scheme 4.To a stirred solution of ω-(+)-biotinoyl-(L)-lysine (50 mg, 0.134 mmol)and NaHCO₃ (23 mg, 0.268 mmol) in water (2 mL) 10 (100 mg, 0.201 mmol)is added. The suspension is diluted with dioxane/dimethylformamide (1/1,4 mL) and stirred at room temperature under an atmosphere of argon for 6d to result in a slightly turbid solution, which is further diluted withwater (10 mL) and lyophilized to give a yellowish powder. The rawmaterial is purified via preparative RP18-HPLC employing a linear 0.1MTEAA (pH 7.0)/CH3CN-gradient (90:10®10:90). Product-containing fractionsare pooled, diluted with water and lyophilized to give 99 mg (86%) of 11as a white powder (the purity of which is larger than 98%, as determinedby analytical RP18-HPLC; UV/fluorescence detection): mp 45° C.;

¹H-NMR (DMSO-d₆): 1.00 (t, 9H, (CH ₃CH₂)₃NH⁺); 1.22-1.63 (m, 11H);1.72-1.88 (m, 2H); 2.03 (t, 2H); 2.55 (d, 1H); 2.62 (q, 6H, (CH₃CH₂)₃NH⁺); 2.77 (dxd, 1H); 3.00-3.07 (m, 2H); 4.07-4.10 (m, 1H); 4.25-4.30(m, 2H, —O—CH ₂—CH═CH₂); 5.30-5.33 (m, 1H, ═CH ₂); 5.43-5.47 (m, 1H, ═CH₂); 6.06-6.13 (m, 1H, —CH═CH₂); 6.32 (s, 1H, NH, biotin); 6.40 (s, 1H,NH, biotin); 7.45 (dxd, J₁=J₂, 1H); 7.63 (dxd, J₁=J₂, 1H); 7.76 (t, 1H,—CONH—(CH₂)₄—, lysine); 7.99, 8.00 and 8.13, 8.14 (AA′BB′, 4H); 8.02 (d,J=5.5, 1H); 8.17, 8.19 and 8.26, 8.28 (AA′BB′, 4H); 8.27 (d, J=6.6, 1H);8.44 (d, J=7.4, 1H, —NH—). ¹³C-NMR (DMSO-d₆): 10.8 ((CH₃CH₂)₃NH⁺); 23.3(CH₂); 25.5 (CH ₂); 28.2 (CH₂); 28.4 (CH₂); 29.2(CH₂); 31.2 (CH₂); 35.4(CH₂); 38.5 (CH₂); 45.6 ((CH₃ CH₂)₃NH⁺); 53.7 (CH); 55.6 (CH); 59.3(CH); 61.2 (CH); 65.4 (—O—CH2-); 111.6 (CH); 118.2 (═CH2); 121.6 (CH);122.0 (CH); 122.8 (C-quart.); 123.4 (CH); 127.7 (CH); 128.4 (CH); 129.1(CH); 129.4 (C-quart.); 130.2 (CH); 132.7 (C-quart.); 132.8 (CH); 137.1(C-quart.); 140.0 (C-quart.); 141.5 (C-quart.); 144.5 (C-quart.); 162.9(C═O); 165.2 (C═O); 165.3 (C═O); 172.0 (C═O); 174.2 (C═O). MS: m/e 753(MH⁺), 775 ([M+Na]⁺). Microanalysis: (C₄₆H₅₉N₇O₇S×2.5 H₂O) calculated,C, 61.45; H, 7.18; N, 10.90; S, 3.56; found, C, 61.64; H, 6.35; N,10.76; S, 3.14.

(c) 4-[3-(4-Allyloxycarbonylphenyl)-1H-indazol-1-yl]-benzoic Acid (5S)3-(Biotinoylamino)-propyl Ester (9) as illustrated in Scheme 4. To astirred solution of 4e (1070 mg, 2.677 mmol), BOP (1303 mg, 2.945 mmol)and DIEA (688 μL, 4.016 mmol) in DMPU (15 mL), a solution of (+)-biotin3-hydroxypropyl amide (807 mg, 2.677 mmol) and 1,2,4-triazole (203 mg,2.945 mmol) in DMPU (10 mL) is slowly injected through a septum at roomtemperature under an atmosphere of argon. The resulting yellow solutionis further stirred under argon for 6 d and evaporated to dryness (HV,bath temperature: 75° C.). The resulting yellow oil is purified by MPLC(dichloromethane:methanol=20:1) to give a yellowish solid (1.258 g,69%), which is further purified by recrystallization fromacetonitrile-water (1:1) yielding 820 mg (45%) of 9: mp 154-157° C.(acetonitrile/water); homogeneous as indicated by thin layerchromatography: R_(f)=0.24 dichloromethane:methanol=15:1). ¹H-NMR(DMSO-d₆): 1.25-1.38 (m, 2H, biotin); 1.42-1.57 (m, 3H, biotin);1.58-1.63 (m, 1H, biotin); 1.88 (txt, J=5.6, 2H, —OCH₂—CH ₂—CH₂NH—);2.08 (t, 2H, —CO—CH ₂—); 2.56 (d, 1H, biotin); 2.79 (dxd, 1H, biotin);3.05-3.10 (m, 1H, biotin); 3.22-3.26 (m. 2H, —OCH₂CH₂—CH ₂—NH—);4.10-4.13 (m, 1H, biotin); 4.27-4.30 (m, 1H, biotin); 4.32 (t, 2H, —O—CH₂—CH₂CH₂NH—); 4.86-4.88 (m, 2H, —O—CH ₂—CH═CH₂); 5.30-5.33 (m, 1H, ═CH₂); 5.42-5.47 (m,1H, ═CH ₂); 6.05-6.13 (m, 1H, —CH═CH₂); 6.34 (s, 1H,NH, biotin); 6.41 (s, 1H, NH, biotin); 7.44 (t, 1H); 7.64 (t, 1H); 7.90(t, 1H, NH—(CH₂)₃—O—); 8.03-8.08 (m, 3H); 8.17-8.21 (m, 4H); 8.24-8.27(m, 3H). ¹³C-NMR-(DMSO-d₆): 25.5; 28.2; 28.4; 28.6; 35.4; 35.5; 40.0;55.6; 59.4; 61.2; 62.9; 65.4; 111.6; 118.2; 121.7; 122.0; 123.0(C-quart.); 123.6; 127.7; 127.8 (C-quart.); 128.5; 129.6 (C-quart.);130.1; 131.0; 132.8; 136.9 (C-quart.); 139.9 (C-quart.); 143.2(C-quart.); 145.0 (C-quart.); 162.9; 165.3; 172.3. MS: m/e 682 (MH⁺).Microanalysis: (C₃₇H₃₉N₅O₆S×H₂O) calculated, C, 63.50; H, 5.90; N,10.01; S, 4.57; found, C, 64.00; H, 5.73; N, 10.42; S, 4.24.

EXAMPLE 5B Synthesis of4-[3-(4-Aminocarbonyl-phenyl)-1H-indazol-1-yl)-benzoic Acid Amides (12a-j) as Illustrated in Scheme 5

Aminoethyl-Tentagel resin (capacity: 0.29 mmol/g) is loaded withphotolabile 4-bromomethyl-3-nitro benzoic acid linker by standardprocedure. Portions of the resin (200 mg; load: 0.27 mmol/g) are reactedwith different primary amines (a-j) (1.08 mmol in 5 mL oftetrahydrofuran) for 24 h at room temperature. The resins are washedwith several portions of tetrahydrofuran, methanol, finally withdichloromethane, and dried in a desiccator under reduced pressure. To asolution of 4c (579 mg, 1.62 mmol) and 1-hydroxybenzotriazole (657 mg,4.86 mmol) in dimethylformamide (30 mL) is addedN,N′-diisopropylcarbodiimide (1.23 g; 9.72 mmol). The reaction mixtureis stirred for 30 min at room temperature. The stock solution is dividedin ten aliquots (3 mL) which are added to the resins prepared. Theresins are shaken at room temperature for 24 h. Reagent containingsolutions are removed by vacuum filtration. The resins are washed withseveral portions of dimethylformamide, methanol, and finally withdichloromethane. The resins are transferred into glass vials and coveredby dichloromethane (5 mL). Methanol is added to achieve isodensesolutions. The vials are shaken under irradiation with an UV-lamp (366nm) for 24 h. After this time the resins are removed by vacuumfiltration and the solvents evaporated to dryness. The purity of theproducts (12 a-j) is analyzed by RP-HPLC, and the presence of thecompounds 12 a-j is verified by mass spectrometry: Yield [%] 12 R HPLC[%] MS (ESI) a

38 69 513 (M + Na)⁺ b

52 76 517 (M + Na)⁺ c

32 64 470 (M + Na)⁺ d

31 58 470 (M + Na)⁺ e

65 77 521 (MH)⁺ f (CH₃)₂CH 23 61 399 (MH)⁺ g CH₃(CH₂)₄ 52 76 427 (MH)⁺ h(CH₃)₂CH(CH₂)₂ 42 89 449 (M + Na)⁺ i

36 63 453 (MH)⁺ j

55 83 433 (M + Na)⁺

EXAMPLE 6B Synthesis of4-(3-{4-[3-(arylsulfonylamino)-propylcarbamoyl]-phenyl}-indazol-1-yl)-benzoicAcid Amides (13 a-d) as illustrated in Scheme 6

Rink amide resin (500 mg; 0.28 mmol) is shaken with piperidine (20%) indichloromethane (10 mL) at room temperature for 1 h. The resin is washedwith portions of dichloromethane, methanol and finally dichloromethane,until a sample of the latest filtrate shows no presence of cleavedprotecting group. The resin is shaken in a solution prepared from 6(0.56 mmol), 1-hydroxybenzotriazole (1.68 mmol), N,N′-diisopropylcarbodiimide (2.24 mmol) and dimethylformamide (35 mL) overnight at roomtemperature. The resin is washed thoroughly with several portions ofdimethylformamide, then methanol, and finally dichloromethane. Thewashing continues until the latest filtrate is free of fluorescence.Solvents are evaporated under reduced pressure at room temperature. TheFmoc protecting group of resin bound 6 is removed as described above fordeprotection of the Rink amide resin. The resin is dried for 30 minunder reduced pressure. Portions of the resin (80 mg, each) are slightlystirred in dichloromethane (5 mL) containing triethylamine (10% v/v).Different arylsulfonylchlorides (a-d; 0.16 mmol, each) are added at roomtemperature. After 3 h the reagents are filtered off, and the resins arewashed with several portions of dichloromethane, methanol, finally withdichloromethane. After drying in a desiccator under reduced pressure,each resin is treated with 3 mL of dichloromethane/trifluoroacetic acid(4/1) for cleavage of the products. The cleavage solutions are collectedby filtration, and the resins are washed with three portions ofdichloromethane (2 mL, each). Solvents are removed under reducedpressure in a desiccator containing sodium hydroxide. The purity of theproducts (13a-d) is analyzed by RP-HPLC, and the presence of thecompounds 13a-d is verified by mass spectrometry: Yield [%] 13 Aryl HPLC[%] MS (ESI) a

31 92 568 (MH)⁺ b

57 85 647 (MH)⁺ c

57 85 599 (MH)⁺ d

79 87 584 (MH)⁺

EXAMPLE 7B4-[3-(4-Carbamoyl-phenyl)-indazol-1-yl]-N-[2-(cyclopropylmethyl-carbamoyl)-ethyl]-benzamide(14) as illustrated in Scheme 7

Aminoethyl-Tentagel resin (capacity: 0.29 mmol/g) is loaded withphotolabile 4-bromomethyl-3-nitro benzoic acid linker by standardprocedure and subsequently reacted with cyclopropylmethylamine asdescribed in example 5B. To a solution of Fmoc-β-alanine (181 mg, 0.58mmol) and 1-hydroxybenzotriazole (235 mg, 1.74 mmol) in DMF (5 mL) isadded N,N′-diisopropylcarbodiimide (293 mg, 2.32 mmol). The solution isleft for 1 h at room temperature. The cyclopropylmethylamine loadedresin (500 mg) is added and slight stirring is applied for 12 h. Theresin is filtered off and washed with DMF, MeOH, and DCM. The resin isadded to a solution (5 mL) of active ester of 4c prepared as describedin example 5B. The reaction mixture is shaken at room temperatureovernight. The reagents are removed by filtration. The resin is washedwith several portions of DMF, MeOH, and finally with DCM. The resin istransferred into a glass vial and covered by DCM (5 mL). MeOH is addedto achieve isodense solution. The vial is shaken under irradiation withan UV-lamp (366 nm) for 24 h. After this time the resin is removed byvacuum filtration and the solvents evaporated to dryness. The purity ofthe product (14) is analyzed by RP-HPLC, and the presence of thecompound 14 is verified by mass spectrometry: m/e=482 (MH⁺). TABLE 1Absorption Emission Excitation λ_(max) λ_(max) λ_(max) 4a

328 nm ε = 22569 M⁻¹ cm⁻¹ 396 nm (360-450 nm)* 328 nm (300-340 nm)* 4b

334 nm ε = 29152 M⁻¹ cm⁻¹ 396 nm (360-500 nm) 335 nm (300-360 nm) 4c

332 nm ε = 30665 M⁻¹ cm⁻¹ 393 nm (360-500 nm) 334 nm (300-360 nm) 4d

334 nm ε = 33114 M⁻¹ cm⁻¹ 396 nm (360-500 nm) 336 nm (300-360 nm) 4e

335 nm ε = 34134 M⁻¹ cm⁻¹ 398 nm (360-480 nm) 337 nm (310-360 nm) 4f

354 nm ε = 25482 M⁻¹ cm⁻¹ 554 nm (470-670 nm) 368 nm (320-400 nm) 4g

328 nm ε = 29157 M⁻¹ cm⁻¹ 364 nm (350-450 nm) 328 nm (310-350 nm) 4h

327 nm ε = 27650 M⁻¹ cm⁻¹ 439 nm (370-600 nm) 330 nm (300-350 nm) 4i

361 nm ε = 31261 M⁻¹ cm⁻¹ 551 nm (470-670 nm) 374 nm (320-420 nm) 4j

358 nm ε = 24967 M⁻¹ cm⁻¹ 565 nm (490-700 nm) 370 nm (340-420 nm) 4k

328 nm ε = 22622 M⁻¹ cm⁻¹ 524 nm (450-620 nm) 331 nm (320-360 nm) 4l

326 nm ε = 17252 M⁻¹ cm⁻¹ 527 nm (450-620 nm) 331 nm (310-360 nm)Solvent: acetonitrile/2% DMSO; range in parenthesis

TABLE 2 Absorption Emission Excitation λ_(max) λ_(max) λ_(max)  8

331 nm ε = 32158 M⁻¹ cm⁻¹ 394 nm (360-480 nm) 332 nm (310-360 nm) 11

334 nm ε = 34349 M⁻¹ cm⁻¹ 397 nm (370-480 nm) 336 nm (310-360 nm)Solvent: acetonitrile/2% DMSO; range in parenthesis.

REFERENCES

Literature Related to Synthesis of 1,3-diarylindazoles

Elderfield, R. C. in “Heterocyclic Compounds” (1957), ed. Elderfield, R.C., vol 5, John Wiley & Sons, Inc., New York, p. 162.

Dalia Croce, Piero; La Rosa, Concetta. Synthesis (1984), 11, 982-3.

Yan, B.; Gstach, H. Tetrahedron Lett. (1996), 37(46),.8325-8.

Wang, Q.; Jochims, J.; Koehlbrandt, S.; Dahlenburg, L.; Al-Talib, M.;Hamed, A., Ismail, Abd El Hamid. Synthesis (1992), 7, 710-8.

Krishnan, R.; Lang, S. A., Jr.; Lin, Yang I; Wilkinson, R. G. J.Heterocycl. Chem. (1988), 25(2), 447-52.

Matsugo, Seiichi; Saito, Mitsuo; Kato, Yohko; Takamizawa, Akira. Chem.Pharm. Bull. (1984), 32(6), 2146-53.

Matsugo, Seiichi; Takamizawa, Akira. Synthesis (1983), 10, 852.

Theilacker, W.; Raabe, C. Tetrahedron Lett. (1966), 91-92.

Gladstone, W. A. F.; Norman, R. O. C. J. Chem. Soc. (1965), 3048-52.

Fries, K.; Fabel, K.; Eckhardt, H. Justus Liebigs Ann. Chem. (1942),550, 31-49.

Borsche, W.; Scriba, W. Justus Liebigs Ann. Chem. (1939), 540, 83-98.

Huisgen, R.; Seidel, M.; Wallbillich, G.; Knupfer, H. Tetrahedron(1962), 17, 3-29.

Gladstone, W. A. F.; Norman, R. O. C. J. Chem. Soc. (1965), 5177-82.

Huisgen, R.; Knupfer, H.; Sustmann, R.; Walibillich, G.; Weberndorfer,V. Chem. Ber. (1967), 100, 1580-92.

Fries, K.; Tampke, H. Justus Liebigs Ann. Chem. (1927), 454, 303-24.

Pummerer, R.; Buchta, E.; Deimler, E. Chem. Ber. (1951), 84(7), 583-90.

Dennler, E. B.; Frasca, A. R. Tetrahedron (1966), 22, 3131-41.

Gladstone, W. A. F.; Harrison, M. J.; Norman, R. O. C. J. Chem. Soc.(1966), 1781.

Literature for Optical Spectroscopy Related to Indazoles:

Saha, Subit K.; Dogra, Sneh K. J. Photochem. Photobiol., A (1997),110(3), 257-266.

Phaniraj, P.; Mishra, Ashok K.; Dogra, S. K. Indian J. Chem., Sect. A(1985), 24A(11), 913-17.

Mishra, A. K.; Swaminathan, M.; Dogra, S. K. J. Photochem. (1984),26(1), 49-56.

Specificic Literature for Fluorescence and UV-Spectroscopy Used in theExperimental Parts:

Melhuish, W. H. (1961) J. Phys. Chem. 65, 229.

Demas, J. N., & Crosby, G. A. (1971) J. Phys. Chem. 75, 991-1024.

Chen, R. F. (1 972) J. of Research of the Intemational Bureau ofStandards Vol. 76A No. 6, 593-606.

Lakowicz, J. R. (1983, pages 52-93, 112-153,156-185) Principles ofFluorescence Spectroscopy, Plenum Press, New York and London.

Savage, M. D. et al., Avidin-Biotin Chemistry, Pierce Chemical Company,(1992).

Petrich, J. W., Chang, M. C., McDonald, D. B., Fleming, G. R., (1983) J.Am. Chem. Soc. 105, 3824-3832.

Szabo, A. G., Rayner, D. M. (1980) J. Am. Chem. Soc. 102, 554-563.

Pugliese; L., Coda, A., Malcovati, M., Bolognesi, M. (1993) J. Mol.Biol. 231, 698

LEGENDS TO FIGURES (i) FIG. 1

(a) Absorption spectrum, of 25 μM 4e in THF and fluorescence excitationspectrum ( - - - ). The small red shift in the fluorescence excitationspectrum is caused by monochromator deviations between the UV and thefluorescence instrument.

(b) Fluorescence emission, 1 μM in THF, excitation at 342 nm.

(ii) FIG. 2

(a) —(i) Excitation and emission spectra of AIDA-conjugates 11, 9, and8, (in the graphical representation assigned as BLI, BPI, and IPB,respectively), and Avidin-Tryptophan, Avidin Lucifer Yellow andAvidin-BODIPY-FL, showing the overlap between tryptophan, BODIPY-FL andLucifer Yellow emission and excitation, respectively.

(iii) FIG. 3

Upper Panel:

Fluorescence resonance energy transfer and ground state quenching in the11 (BLI) (a) and 8 (IPB) (b) tryptophan avidin complexes. The curves areassigned by numbers 1-5.

The difference spectra [I(11 or 8(free))+I(avidin(free))]−I(11- or8-avidin Complex) are shown in curves 5.

The 11 fluorescence is quenched by about 25.5% by complexation in theavidin binding site. Aproximately 62% of this total quenching effect isfluorescence resonance energy transfer. From 250 to 305 nm theenhancement of the tryptophan and tyrosine fluorescence intensity is 72%based on the sum of 11 and avidin. Linking the biotin to position 3 inthe indazole molecule (from position 1 in 11) not only changes thefluorescence spectra, but also the tryptophan →indazole FRETcharacteristics: Indicated by 26% increase in indazole fluorescenceemission (instead of quenching) an enhanced energy transfer totryptophan (120%) is superimposed by a strong increase in indazolequantum yield in the avidin environment.

Lower Panel:

Fluorescence resonance energy transfer and ground state quenching in the11 (c) and 8 (d)BODIPY-FL avidin complexes. The curves are assigned bynumbers 1-5. With both molecular geometries, the indazole tracer linkedvia position 1 (11) and position 3(8), binding of BODIPY-FL labelledavidin causes a 70-90% quenching of the indazole emission. Thisreduction in signal intensity can be caused by ground state quenching ofthe indazole or FRET to the BODIPY labels on avidin with concurrentnon-radiative depopulation of the excited state. Although the overlapintegral between the indazole emission and the BODIPY-FL absorptionspectrum is smaller for 8 than for 11, more sensitization of acceptorfluorescence occurs in the 8-avidin BODIPY complex resulting in 48%fluoresence energy transfer and 93.5% indazole donor quenching.

This effect can be caused by a higher donor quantum yield or a preferedrelative orientation of the donor emission and acceptor absoprtiondipoles in 8.

1-11. (canceled)
 12. Compounds comprising structures of the formulaeII-IIIA-B-D-C-D′-   (Formula (II))A-B-D- and -D′-C   (Formula (III)) wherein A is selected from the groupconsisting of functionalized polystyrene based resins, polyacrylamidebased polymers, polystyrene/polydimethylacrylamide composites, PEGAresins, polystyrene-polyoxyethylene based supports, Tentagel resins,PEG-polystyrene graft polymeric supports, glass surfaces, functionalizedsurfaces, materials grafted with functionalized surfaces, andpolyethylenglycol; B is a linker allowing cleavage of fluorescentconjugates of formula (II-III) for liberation of the D and C containingfragments; C is a compound selected from formula (I)

wherein one of the radicals R¹ or R² and one of the radicals R³ or R⁴ ishydrogen and the other is independently —COOH, —COOR⁷, —CONH₂,—CONH(CH₂)_(n)OH, —CONR⁸R⁹, —CH₂OH, —CH₂NH₂, —NO₂, —NR¹⁰R¹¹, —NHCOR¹²,Cl, Br, F, —CF₃, —N═C═O, —N═C═S, —SO₃H, —SO₂NH(CH₂)_(n)NH₂, (C₁-C₄)alkyl, (C₁-C₁₆)-alkyl substituted at the terminal carbon with —COOH,—COOR⁷, —CONH₂, —CONR⁸R⁹, —CONH(CH₂)_(n)OH, —CH₂OH, —CH₂NH₂, —N═C═O,—N═C═S, —SO₃H, —SO₂NH(CH₂)_(n)NH₂, —CONH(CH₂)_(n)NH₂, and the —NH₂ groupcould also be substituted by (C₁-C₄) alkyl or a commonly used aminoprotecting group; and one of the radicals R⁵ or R⁶ is hydrogen and theother is hydrogen, halogen, —NO₂, —NR¹⁰R¹¹, —NHCOR¹², (C₁-C₄) alkyl,(C₁-C₁₆)-alkyl substituted at the terminal carbon with —COOH, —COOR⁷,—CONH₂, —CONR⁸R⁹, —CONH(CH₂)_(n)OH, —CH₂OH, —CH₂NH₂, —N═C═O, —N═C═S,—SO₃H, —SO₂NH(CH₂)_(n)NH₂, —CONH(CH₂)_(n)NH₂, wherein and the —NH₂ groupcould also be substituted by (C₁-C₄) alkyl or a commonly used aminoprotecting group; n is 2-8; with the proviso that only one of R¹-R⁶ isnitro; R⁷ is a commonly used carboxyl protecting or carboxyl activatinggroup; R⁸ or R⁹ is hydrogen and the other is lower alkyl (C₁-C₄),phenyl, benzyl, or R⁸ and R⁹ are part of a 5 or 6 membered ring; R¹⁰ andR¹¹ are independently hydrogen or (C₁-C₄)alkyl; and R¹² is(C₁-C₁₀)alkyl, phenyl, which both can be substituted by (C₁-C₄) alkyl,protected amino group or halogen; and D and D′ are independently a bondor a spacer selected from α,ω-diamino-alkanes, diaminocyclohexyl,bis-(aminomethyl)-substituted phenyl, α-amino-ω-hydroxy-alkanes,alkylamines, cyclic alkylamines or cyclic alkyldiamines or amino acidswithout or with additional functionality in the side chain. 13.Compounds of claim 12, wherein B is selected from benzyl, benzhydryl,benzhydryliden, trityl, xanthenyl, benzoin, silicon, or allyl basedlinkers.
 14. Compounds of claim 12 of the following structures:


15. Compounds of claim 12 wherein the amino protecting group istert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, phthalimido,trifluoroacetamido, methoxycarbonyl, ethoxycarbonyl, benzyloxycarbonyl,allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, or2-(trimethylsilyl)ethoxy-carbonyl.
 16. Compounds of claim 12 wherein Cis of the following structures: